Humanin-like peptide and use thereof

ABSTRACT

A novel polypeptide having a cell death inhibitory activity and use thereof is provided. The polypeptide and the polynucleotide encoding it can be used as a diagnostic, therapeutic or prophylactic agent for various diseases and disorders. Certain suitable diseases and disorders which may be diagnosed, treated, or prevented with the polypeptide and the polynucleotide encoding it are selected from neurodegenerative diseases, brain dysfunctions, cancers, immunological disease, infections, gastrointestinal diseases, circulatory diseases, and endocrine diseases. The polypeptide and the polynucleotide encoding it can be used as a cell death inhibitor.

TECHNICAL FIELD

The present invention relates to a novel polypeptide regulatingbiological functions, a DNA encoding the polypeptide and so on. Morespecifically, the present invention relates to prophylactics and/ortherapeutics or diagnostics agents for neurodegenerative diseases, braindysfunctions, etc.

BACKGROUND ART

Alzheimer's disease is a representative neurodegenerative diseaseaccompanied by progressive dementia and loss of cognitive ability,however effective treatment method for this disease has not been found.Alzheimer's disease is obviously one of the most serious diseases inaging societies at present, and thus development of therapeutics forthis disease is extremely significant from the viewpoint of medicaleconomy.

Recently, Hashimoto et al., paid attention to the fact that there areless lesions in the occipital lobe of Alzheimer's disease patients andhave cloned a gene by using the “death-trap” method (L. D'Adamio et al.,Semin. Immunol., 9:17–23, 1997) from the occipital lobe that inhibitsthe death of nerve cells into which the causative gene of familialAlzheimer's disease is introduced (Proc. Natl. Acad. Sci. USA 98:6336–6341, 2001). This gene encodes a peptide designated “humaitin” (WO01/21787) consisting of 24 residues. A synthetic humanin peptide notonly inhibited the death of nerve cells into which the causative gene offamilial Alzheimer's disease is introduced, but also the death of nervecells induced by the addition of β amyloid which is considered to be apotential cause for Alzheimer's disease. These findings suggest thathumanin or derivatives thereof might be usable as therapeutic agents forAlzheimer's disease.

Although Alzheimer's disease is a representative neurodegenerativedisease accompanied by progressive dementia and loss of cognitiveability, however, effective treatment method has been not found untilnow.

DISCLOSURE OF THE INVENTION

The present inventors have made intensive and extensive researchesdirecting their attention to these facts. As a result, the presentinventors have expected the presence of a humanin-like gene in humangenome and found a humanin-like sequence by searching through a humangenome database (GEMBLE) using the humanin gene sequence. Further, basedon this sequence information, the inventors have succeeded in cloning agene encoding a humanin-like peptide from a human brain cDNA library.Like humanin, this newly found peptide consisted of 24 residues, ofwhich 6 residues were different from those of humanin. The presentinventors have also found that this peptide has an inhibitory effect onthe death of nerve cells. As a result of further researches based onthese findings, the present invention has been achieved.

The present invention relates to:

-   (1) A polypeptide comprising an amino acid sequence identical or    substantially identical with the amino acid sequence as shown in SEQ    ID NO: 4, or an amide, ester or salt thereof;-   (2) The polypeptide according to (1) above consisting of the amino    acid sequence as shown in SEQ ID NO: 4, or an amide, ester or salt    thereof;-   (3) A partial peptide of the polypeptide according to (1) above, or    amide, ester or salt of the partial peptide;-   (4) A polynucleotide comprising a polynucleotide having a nucleotide    sequence encoding the polypeptide according to (1) above;-   (5) The polynucleotide according to (4) above, wherein the    polynucleotide is DNA;-   (6) The polynucleotide according to (4) above comprising a    nucleotide having the nucleotide sequence as shown in SEQ ID NO: 3;-   (7) A polynucleotide comprising a polynucleotide having a nucleotide    sequence encoding the partial peptide according to (3) above;-   (8) A recombinant vector comprising the polynucleotide according    to (4) or (7) above;-   (9) A transformant transformed with the recombinant vector according    to (8) above;-   (10) A method for producing tie polypeptide according to (1) above    or an amide, ester or salt thereof or the partial peptide according    to (3) above or an amide, ester or salt thereof, comprising    culturing the transformant according to (9) above and allowing the    polypeptide according to (1) above or the partial peptide according    to (3) above to be produced and accumulated;-   (11) An antibody to the polypeptide according to (1) above or an    amide, ester or salt thereof, or to the partial peptide according    to (3) above or an amide, ester or salt thereof;-   (12) A polynucleotide having a nucleotide sequence, or a part    thereof, complementary to or substantially complementary to the    polynucleotide encoding a polypeptide, or a part thereof, comprising    an amino acid sequence identical with or substantially identical    with the amino acid sequence as shown in SEQ ID NO: 4;-   (13) A method for screening for compounds, or salts thereof, that    promote or inhibit the activity of the polypeptide according to (1)    above or an amide, ester or salt thereof, or the partial peptide    according to (3) above or an amide, ester or salt thereof, wherein    the method is characterized by using the polypeptide according    to (1) above, or an amide, ester or salt thereof, or the partial    peptide according to (3) above or an amide, ester or salt thereof;-   (14) A kit for screening for compounds, or salts thereof, that    promote or inhibit the activity of the polypeptide according to (1)    above or an amide, ester or salt thereof, or the partial peptide    according to (3) above or an amide, ester or salt thereof, which    comprises the polypeptide according to (1) above or an amide, ester    or salt thereof, or the partial peptide according to (3) above or an    amide, ester or salt thereof;-   (15) A compound, or a salt thereof, that promotes the activity of    the polypeptide according to (1) above or an amide, ester or salt    thereof, or the partial peptide according to (3) above or an amide,    ester or salt thereof, which is obtainable by using the screening    method according to (13) above or the screening kit according    to (14) above;-   (16) A compound, or a salt thereof, that inhibits the activity of    the polypeptide according to (1) above or an amide, ester or salt    thereof, or the partial peptide according to (3) above or an amide,    ester or salt thereof, which is obtainable by using the screening    method according to (13) above or the screening kit according    to (14) above;-   (17) A medicine comprising the compound according to (15) or a salt    thereof,-   (18) A medicine comprising the compound according to (16) or a salt    thereof,-   (19) A medicine comprising the polypeptide according to (1) above or    an amide, ester or salt thereof, or the partial peptide according    to (3) above or an amide, ester or salt thereof,-   (20) A medicine comprising the polynucleotide according to (4)    above;-   (21) A diagnostic agent comprising the polynucleotide according    to (4) above;-   (22) A medicine comprising the antibody according to (11) above;-   (23) A diagnostic agent comprising the antibody according to (11)    above;-   (24) A medicine comprising the polynucleotide according to (12)    above;-   (25) A diagnostic agent comprising the polynucleotide according    to (12) above;-   (26) The medicine according to (17), (19) or (20) above, wherein the    medicine is a prophylactic and/or therapeutic agent for    neurodegenerative disorders or brain dysfunctions;-   (27) The medicine according to (26) above, wherein the medicine is a    prophylactic and/or therapeutic agent for Alzheimer's disease,    Parkinson's disease, Down syndrome, amyotrophic lateral sclerosis,    prion disease, Creutzfeldt-Jacob disease, Huntington's chorea,    diabetic neuropathy, multiple sclerosis, brain infarction, cerebral    hemorrhage, subaraclinoid hemorrhage, ischemic brain diseases,    epidural hematoma or subdural hematoma;-   (28) The medicine according to (26) above, wherein the medicine is a    prophylactic and/or therapeutic agent for Alzheimer's disease;-   (29) The medicine according to (17), (19) or (20) above, wherein the    medicine is a cell death inhibitor;-   (30) The diagnostic agent according to (21), (23) or (25) above,    which is a diagnostic agent for diseases accompanied by    neurodegeneration;-   (31) The diagnostic agent according to (30) above, which is a    diagnostic agent for Alzheimer's disease, Parkinson's disease, Down    syndrome, amyotrophic lateral sclerosis, prion disease,    Creutzfeldt-Jacob disease, Huntington's chorea, diabetic neuropathy,    multiple sclerosis, brain infarction, cerebral hemorrhage,    subarachnoid hemorrhage, ischemic brain diseases, epidural hematoma    or subdural hematoma;-   (32) A method of preventing and/or treating neurodegenerative    diseases or brain dysfunctions, which is characterized by    administering to a mammal an effective amount of the polypeptide    according to (1) above or an amide, ester or salt thereof, or the    partial peptide according to (3) above or an amide, ester or salt    thereof;-   (33) A method of preventing and/or treating neurodegenerative    diseases or brain dysfunctions, which is characterized by    administering to a mammal an effective amount of the polynucleotide    according to (4) above;-   (34) A method of preventing and/or treating neurodegenerative    diseases or brain dysfunctions, which is characterized by    administering to a mammal an effective amount of the compound    according to (15) above or a salt thereof;-   (35) Use of the polypeptide according to (1) above or an amide,    ester or salt thereof or the partial peptide according to (3) above    or an amide, ester or salt thereof, for producing a prophylactic    and/or therapeutic agent for neurodegenerative diseases or brain    dysfunctions;-   (36) Use of the polynucleotide according to (4) above for producing    a prophylactic and/or therapeutic agent for neurodegenerative    diseases or brain dysfunctions;-   (37) Use of the compound according to (15) above or a salt thereof,    for producing a prophylactic and/or therapeutic agent for    neurodegenerative diseases or brain dysfunctions.

Further, the polypeptide, the partial peptide, the nucleotide (e.g. DNA)and so forth of the invention may be applicable to molecular markers,tissue markers, chromosome mapping, identification of genetic diseases,diagnosis of disease states, or basic researches such as designing ofprimers or probes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the inhibitory effect of various concentrations of thehumanin-like peptide upon glutamic acid-induced cell death of ratadrenal medulla-derived pheochromocytoma cell PC12h. Cell survivalratios are shown taking the survival in the glutamic acid not-added plotas 100%. Mark * represents a significant difference (p<0.05) compared tothe humanin-like peptide not-added plot.

BEST MODE FOR CARRYING OUT THE INVENTION

The polypeptide of the invention comprising an amino acid sequenceidentical or substantially identical with the amino acid sequence asshown in SEQ ID NO: 4 (hereinafter, sometimes referred to as the“polypeptide of the invention”; sometimes, a polypeptide comprising anamino acid sequence identical or substantially identical with the aminoacid sequence as shown in SEQ ID NO: 4 or an amide, ester or saltthereof may also be called the “polypeptide of the invention”collectively) may be a polypeptide derived from cells of any kind (e.g.hepatocytes, splenocytes, nerve cells, glia cells, pancreatic β cells,bone marrow cells, mesangial cells, Langerhan's cells, epidemial cells,epithelial cells, endothelial cells, fibroblasts, fibrous cells, musclecells, fat cells, immune cells (e.g. macrophages, T cells, B cells,natural killer cells, mast cells, neutrophils, basophils, eosinophils,monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes,osteoblasts, osteoclasts, mammary cells or interstitial cells, orprogenitor cells of these cells, stem cells or cancer cells, etc.) ofhuman or other warm-blooded animals (e.g. guinea pig, rat, mouse,chicken, rabbit, pig, sheep, bovine, monkey, etc.) or any tissue inwhich such cells are present, such as brain, various parts of brain(e.g. olfactory bulb, amygdaloid nucleus, cerebral basal nucleus,hippocampus, thalamus, hypothalamus, cerebral cortex, medulla oblongata,cerebellum), spinal cord, pituitary gland, stomach, pancreas, kidney,liver, gonad, thyroid, gall-bladder, bone marrow, adrenal gland, skin,muscle, lung, gastrointestinal tracts (e.g. large intestine, smallintestine), blood vessels, heart, thymus, spleen, salivary gland,peripheral blood, prostate, testis, ovary, placenta, uterus, bone,cartilage, joint, skeletal muscle, etc. It may also be a recombinantpolypeptide or a synthetic polypeptide.

The term “substantially identical” means that the activity of thepolypeptide, for example, cell death inhibitory effect (e.g. inhibitoryeffect against cell death associated with various diseases), cellsurvival maintaining effect, or prophylactic and/or therapeutic activity(effect) upon neurodegenerative diseases, cancers, immunologicaldiseases, infections, gastrointestinal diseases, circulatory diseases,endocrine diseases, etc. or physiological characteristics of thepolypeptide are substantially identical. As long as substitution,deletion, addition or insertion of amino acids does not cause asignificant change in physiological properties or chemical properties ofa polypeptide, the polypeptide that has undergone the substitution,deletion, addition or insertion may be said substantially identical withthe initial polypeptide that has not undergone such substitution,deletion, addition or insertion. The substantially identical substitutedamino acid in the above amino acid sequence may be selected from, forexample, the other amino acids in the class to which the initial aminoacid belongs.

Examples of non-polar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan andmethioine. Examples of polar (neutral) amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine and glutamine.Examples of positively charged (basic) amino acids include arginine,lysine and histidine. Examples of negatively charged (acidic) aminoacids include aspartic acid and glutamic acid.

An amino acid sequence substantially identical wit the amino acidsequence as shown in SEQ ID NO: 4 is not particularly limited as long asthe polypeptide comprising the relevant amino acid sequence has anactivity (nature) substantially identical with the activity (nature) ofa polypeptide comprising the amino acid sequence as shown in SEQ ID NO:4. For example, an amino acid sequence having about 80% or more,preferably about 85% or more, more preferably about 90% or more, mostpreferably about 95% or more homology to the amino acid sequence asshown in SEQ ID NO: 4 may be used.

Examples of the activity (nature) of substantially the same qualitymentioned above include an activity (effect) that is qualitativelyidentical to the cell death inhibitory effect (e.g. inhibitory effectagainst cell death associated with various diseases), cell survivalmaintaining effect, or prophylactic and/or therapeutic activity (effect)upon neurodegenerative diseases, cancers, immunological diseases,infections, gastrointestinal diseases, circulatory diseases, endocrinediseases, etc. possessed by a polypeptide comprising the amino acidsequence as shown in SEQ ID NO: 4.

More specific examples of polypeptides comprising the amino acidsequence as shown in SEQ ID NO: 4 include the so-called muteins, such aspolypeptides comprising (i) the amino acid sequence of SEQ ID NO: 4wherein one or two or more amino acids (preferably about 1–6, morepreferably about 1–3, and still more preferably 1 or 2 amino acids) aredeleted therefrom; (ii) the amino acid sequence of SEQ ID NO: 4 whereinone or two or more amino acids (preferably about 1–6, more preferablyabout 1–3, still more preferably 1 or 2 amino acids) are added thereto;(iii) the amino acid sequence of SEQ ID NO: 4 wherein one or two or moreamino acids (preferably about 1–6, more preferably about 1–3, still morepreferably 1 or 2 amino acids) are replaced with other amino acids; or(iv) an amino acid sequence which is a combination of these sequences.

When the amino acid sequence is inserted, deleted or substituted asdescribed above, the position of insertion, deletion or substitution isnot particularly limited.

As a partial peptide of the polypeptide of the invention (i.e. thepartial peptide of the invention), any partial peptide of theabove-described polypeptide of the invention may be used. For example, apartial peptide having an activity of substantially the same quality asthat of the polypeptide of the invention is preferably used (the term“activity of substantially the same quality” has the same meaning asdescribed above).

However, being different from the polypeptide of the invention, thepartial peptide of the invention may be used as antigen for preparingantibodies. Thus, the partial peptide of the invention does notnecessarily need to have the activity possessed by the polypeptide ofthe invention.

More specific examples of the partial peptide of the invention include apartial peptide of the polypeptide of the invention comprising an aminoacid sequence identical with or substantially identical with the aminoacid sequence as shown in SEQ ID NO: 4.

The term “substantially identical” has the same meaning as defined abovein the description of the polypeptide of the invention.

More specific examples of partial peptides comprising an amino acidsequence substantially identical with the amino acid sequence as shownus SEQ ID NO: 4 include the so-called muteins, such as partial peptidescomprising (i) the amino acid sequence of SEQ ID NO: 4 wherein one ortwo or more amino acids (e.g. about 1–20, preferably about 1–15,preferably about 1–10, preferably about 1–5, and more preferably 1 or 2amino acids) are deleted therefrom; (ii) the amino acid sequence of SEQID NO: 4 wherein one or two or more amino acids (e.g. about 1–20,preferably about 1–15, preferably about 1–10, preferably about 1–5, morepreferably 1 or 2 amino acids) are added thereto; (iii) the amino acidsequence of SEQ ID NO: 4 wherein one or two or more amino acids (e.g.about 1–5, more preferably 1 or 2 amino acids) are replaced with otheramino acids; or (iv) an amino acid sequence which is a combination ofthese sequences.

When the amino acid sequence is inserted, deleted or substituted asdescribed above, the position of insertion, deletion or substitution isnot particularly limited.

Specific examples of the partial peptide of the invention include apeptide having an amino acid sequence spanning from position 19 toposition 24 (SEQ ID NO: 7), from position 5 to position 24, fromposition 1 to position 20, from position 5 to position 20, from position1 to position 21 or from position 5 to position 21 of the amino acidsequence as shown in SEQ ID NO: 4.

The polypeptide and the partial peptide of the invention also encompassthose polypeptides/peptides where substituents on side chains ofintramolecular amino acids are protected with appropriate protectivegroups, or conjugated peptides such as glycopeptides to which sugarchains are attached.

Further, the polypeptide of the invention or the peptide of theinvention may exist not only as a monomer but also as a dimer, a trimer,a tetramer, etc. Specifically, possible forms include, but are notlimited to, the following: two polypeptides of the invention form adimer; or two partial peptides of the invention form a dimer; or thepolypeptide of the invention and the partial peptide of the inventionform a dimer.

Further, the polypeptide and the partial peptide of the invention maycomprise any foreign sequence (for example, such as FLAG, His tag, HAtag or HSV tag) that could be an epitope (antibody recognition site)located at, for example, their N-terminal or C-terminal.

The polypeptide and the partial peptide of the invention are expressedin accordance with the conventions for description of peptides, that is,the N-terminus (amino terminus) at the left end and the C-terminus(carboxyl terminus) at the right end. The C-terminus of the polypeptideof the invention (such as a polypeptide comprising the amino acidsequence as shown in SEQ ID NO: 4) may be a carboxyl group (—COOH), acarboxylate (—COO), an amide (—CONH₂) or an ester (—COOR).

Examples of R of the above ester group include C₁₋₆ alkyl groups (e.g.methyl, ethyl, n-propyl, isopropyl or n-butyl), C₃₋₈ cycloalkyl groups(e.g. cyclopentyl or cyclohexyl), C₆₋₁₂ aryl groups (e.g. phenyl orα-naphthyl), C7₋₁₄ aralkyl groups such as phenyl-C₁₋₂ alkyl groups (e.g.benzyl or phenethyl) and α-naphthyl-C₁₋₂ alkyl groups (e.g.α-naphthylmethyl). In addition, the ester group also includespivaloyloxynethyl esters that are universally used as oral esters.

When the polypeptide or the partial peptide of the invention has acarboxyl group (or carboxylate) at any position other than itsC-terminus, the carboxyl group may be amidated or esterified; such apolypeptide or partial peptide is also included in the polypeptide orthe partial peptide of the invention. The ester in this case may be, forexample, any of the esters mentioned above for the C-terminal ester.

Furthermore, the polypeptide or the partial peptide of the inventionincludes those polypeptides or partial peptides in which the N-terminalamino acid residue (e.g. Met) is protected by a protective group (e.g.C₁₋₆ acyl group such as C₁₋₆ alkanoyl group (e.g. formyl group or acetylgroup)); those polypeptides in which the N-terminal Glu generatedthrough in vivo cleavage is pyroglutaminated; those polypeptides,peptides or partial peptides in which a substituent on a side chain ofan amino acid (e.g. —OH, —SH, amino group, imidazole group, indolegroup, or guannidino group) is protected by an appropriate protectivegroup (e.g. C₁₋₆ acyl group such as C₁₋₆ alkanoyl group (e.g. formylgroup or acetyl group)); and conjugated polypeptides such as theso-called glycopolypeptides to which sugar chains are linked.

As the salt of the polypeptide or the partial peptide of the invention,salts formed with physiologically acceptable acids (e.g. organic orinorganic acids) or bases (e.g. alkali metals) are used. Especiallypreferable are physiologically acceptable acid addition salts. Examplesof such salts include salts formed with inorganic acids. (e.g.hydrochloric acid, phosphoric acid, hydrobromic acid or sulfuric acid)and salts formed with organic acids (e.g. acetic acid, formic acid,propioinc acid, fumaric acid, maleic acid, succinic acid, tartaric acid,citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acidor benzenesulfonic acid).

The polypeptide or the partial peptide of the invention can be producedfrom the afore-mentioned cells or tissues of human or other warm-bloodedanimals by known purification methods for polypeptides (proteins).Alternatively, the polypeptide or the partial peptide of the inventioncan be produced by culturing a transformant comprising thepolynucleotide (DNA, etc.) of the invention described later encoding thepolypeptide or the partial peptide of the invention. It can also beproduced in accordance with the procedures for peptide synthesis whichare described later.

When the polypeptide or the partial peptide of the invention is producedfrom tissues or cells of human or non-human mammals, the relevant tissueor cell is homogenized and then the polypeptide of the present inventionis extracted with acids, etc. The polypeptide or the partial peptide canbe purified and isolated from the resultant extract by a combination ofchromatography, such as reversed phase chromatography, ion exchangechromatography and so on.

For the synthesis of the polypeptide or the partial peptide of theinvention or a salt or amide thereof, any of the commercial resinsavailable for polypeptide (protein) synthesis may be used. Examples ofsuch resins include chloromethyl resin, hydroxymethyl resin,benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcoholresin, 4-methylbenzhydrylamine resin, PAM resin,4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenylhydroxymethyl)phenoxy resin, and4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)phenoxy resin. Using such aresin, amino acids protected at their α-amino groups and side chainfunctional groups are condensed on the resin according to the amino acidsequence of the polypeptide of interest by conventional condensationmethods. At the final stage of the reaction, all protective groups areremoved simultaneously with the cleavage of the polypeptide from theresin. Then, in a highly diluted solution, intramolecular disulfide bondformation reaction is carried out to obtain the polypeptide of interestor amide thereof.

With respect to the condensation of the above-described protected aminoacids, various activators may be useful for polypeptide synthesis, amongall, carbodiimide reagents are especially preferred. Carbodiimidereagents include DCC, N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminoprolyl)carbodiimide. For activation by thesereagents, protected amino acids may be directly added to the resin witha racemization inhibitor addictive such as HOBt or HOOBt, or protectedamino acids may be added to the resin after the protected amino acidsmay be activated as a corresponding acid anhydride or HOBt ester orHOOBt ester.

The solvent used for the above-mentioned activation of protected aminoacids or the condensation thereof with a resin may be appropriatelyselected from those solvents known to be useful for polypeptide(protein) condensation reactions. Useful solvents include acid amides(e.g. N,N-dimethylformamide, N,N-dimethylacetamide orN-methylpyrrolidone), halogenated hydrocarbons (e.g. methylene chloride,or chloroform), alcohols (e.g. trifluoroethanol), sulfoxides (e.g.dimethyl sulfoxide), ethers (e.g. pylidine, dioxane, tetrahydrofuran),nitrites (e.g. acetonitrile or propionitrile), esters (e.g. methylacetate or ethyl acetate), and suitable mixtures of these solvents. Thereaction temperature may be appropriately selected from the range knownto be useful for polypeptide (protein) bond-forming reactions; usually,the temperature is selected from the range from about −20° C. to about50° C. The activated amino acid derivative is usually used in 1.5- to4-fold excess. When the condensation is found insufficient as a resultof test using the ninhydrin reaction, sufficient condensation can beachieved by repeating reactions without removing protective groups. Whensufficient condensation cannot be achieved even by repeating reactions,unreacted amino acids may be acetylated with acetic anhydride oracetylimidazole so that they do not affect subsequent reactions.

Protective groups for the amino group of raw materials include Z, Boc,t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl,Cl-Z, Br-Z, adamantyloxycarbonyl, trifluoroacetyl, phthaloyl, formyl,2-nitrophenylsulfenyl, diphenylphosphinothioyl, and Fmoc.

The carboxyl group can be protected, for example, in the form of analkyl ester (e.g. straight-chain, branched, or cyclic alkyl esters suchas methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl, and so on); aralkyl ester (e.g.benzyl, 4-nitrobenzyl, 4-methoxybenzyl, 4-chlorobenzyl, benzhydryl, andso on), phenacyl ester, benzyloxycarbonylhydrazide,t-butoxycarbonylhydrazide or tritylhydrazide.

The hydroxyl group of serine can be protected, for example, byesterification or etherification. Examples of suitable groups for thisesterification include lower (C₁₋₆) alkanoyl groups such as acetyl,aroyl groups such as benzoyl, and carbonic acid-derived groups such asbenzyloxycarbonyl and ethyloxycarbonyl. Examples of groups suitable forthe etherification include benzyl, tetrahydropyranyl and t-butyl.

Protective groups for the phenolic hydroxyl group of tyrosine includeBzl, Cl₂-Bzl, 2-nitrobenzyl, BrZ, and t-butyl.

Protective groups for the imidazole ring of listidine include Tos,4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl, Bum,Boc, Trt and Fmoc.

Activated carboxyl groups of raw materials include the correspondingacid anhydrides, azides and active esters (esters of alcohols such aspentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, cyanomethylalcohol, p-nitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimideand HOBt). Examples of raw materials with activated amino groups includethe corresponding phosphoric acid amides.

Methods for removing (eliminating) protective groups include, forexample, catalytic reduction in a hydrogen stream in the presence of acatalyst such as Pd black or Pd-carbon, acid treatment with anhydroushydrogen fluoride, methanesulfonic acid, trifluoromethanesulfonic acid,trifluoroacetic acid or mixtures thereof, treatment with a base such asdiiso-propylethylamine, triethylamine, piperidine, piperazine or thelike, and reduction with sodium in liquid ammonia. The eliminationreaction by the above-mentioned acid treatment is generally conducted attemperatures of about −20° C. to about 40° C. In the acid treatment, itis effective to add a cation scavenger such as anisole, phenol,thioanisole, m-cresol, p-cresol, dimethylsulfide, 1,4-butanedithiol or1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the protectivegroup for the imidazole ring of histidine is removed by thiophenoltreatment. The formyl group used as the protective group for the indolering of tryptophan may be removed by the above-mentioned deprotection bythe acid treatment in the presence of 12-ethanedithiol,1,4-butanedithiol or the like, or by alkali treatment using dilutesodium hydroxide, dilute ammonia or the like.

The protection of functional groups which should not be taken part inthe reaction and protective groups therefor, the removal of theseprotective groups and the activation of functional groups involved inthe reaction can be appropriately selected from groups or methods knownin the art.

An alternative method for obtaining amides of the polypeptide or partialpeptide of the invention comprises, for example, protecting theα-carboxyl group of the C-terminal amino acid by amidation, extendingthe peptide (polypeptide) chain to a desired length on the side of theamino group, preparing a polypeptide with its N-terminal α-amino groupselectively deprotected, preparing a polypeptide with its C-terminalcarboxyl group selectively deprotected, and condensing these twopolypeptides in a mixed solvent such as described above. Details of thiscondensation reaction are the same as described above. Afterpurification of the protected polypeptide thus obtained by condensation,all the protective groups are removed by the method described above tothereby to provide a crude polypeptide of interest. This crudepolypeptide is purified by various known purification techniques andlyophilized to provide the desired polypeptide or partial peptide in anamide form.

A method for obtaining esters of the polypeptide or the partial peptideof the invention, for example, condensing the α-carboxyl group of theC-terminal amino acid with a desired alcohol to prepare thecorresponding amino acid ester, and subjecting this ester to the sameprocedures as described above in the preparation of amides to therebyprovide the desired polypeptide or partial peptide in an ester form.

The polypeptide or the partial peptide of the invention can be producedby known methods for peptide synthesis. The method for peptide synthesismay be solid-phase synthesis or liquid-phase synthesis. Briefly, apeptide of interest can be produced by condensing a partial peptide oramino acids capable of constituting the partial peptide of the inventionwith the residual part thereof and, if the product has protectivegroups, removing the protective groups. Examples of condensation methodsand methods for removal of protective groups known in the art includethose described in the following references (i) to (v).

-   (i) M. Bodanszky & M. A. Ondetti, Peptide Synthesis, Interscience    Publishers, New York, 1966-   (ii) Schroeder & Luebke, The Peptide, Academic Press, New York, 1965-   (iii) Nobuo Izumiya et al., Fundamentals and Experiments in Peptide    Synthesis, Maruzen, 1975-   (iv) Haruaki Yajima and Shumpei Sakakibara, Biochemical Experiment    Series 1, Polypeptide Chemistry IV, 205, 1977, and-   (v) Haruaki Yajima (ed.), Development of Drugs (Continued), Vol. 14,    Peptide Synthesis, Hirokawa Shoten

After the reaction, the polypeptide or the partial peptide of theinvention can be isolated and purified by a combination of conventionalpurification techniques such as solvent extraction, distillation, columnchromatography, liquid chromatography, and recrystallization. When thepolypeptide thus obtained is a free polypeptide, it can be converted toa suitable salt by know methods or methods based thereon. On thecontrary, when the polypeptide is obtained in a salt form, it can beconverted to a free polypeptide or another salt by known methods ormethods based thereon.

The polynucleotide encoding the polypeptide or the partial peptide ofthe invention (hereinafter, such polynucleotide is sometimes referred toas the “polynucleotide of the invention” collectively) may be anypolynucleotide as long as it comprises a nucleotide sequence encodingthe above-described polypeptide or partial peptide of the invention (DNAor RNA; preferably, DNA). The polynucleotide is a DNA or RNA (such asmRNA) encoding the receptor protein of the invention, and may bedouble-stranded or single-stranded. When the polynucleotide isdouble-stranded, it may be a double-stranded DNA, a double-stranded RNA,or a DNA:RNA hybrid. When the polynucleotide is single-stranded, it maybe a sense strand (i.e. coding strand) or an anti-sense strand (i.e.non-coding strand).

The DNA encoding the polypeptide or the partial peptide of the inventionmay be genomic DNA, cDNA derived from the above-mentioned cells ortissues, or synthetic DNA. Vectors used for library construction may beany vectors such as bacteriophage, plasmid, cosmid, phagemid, and so on.Alternatively, total RNA or mRNA fraction may be prepared from theabove-mentioned cells or tissues, followed by direct amplification byreverse transcriptase polymerase chain reaction (hereinafter,abbreviated to as “RT-PCR”).

With respect to the DNA encoding the polypeptide of the invention, theDNA may be any DNA as long as it comprises a DNA encoding a polypeptidehaving an activity (nature) of substantially the same quality as that ofthe polypeptide of the invention [e.g. cell death inhibitory effect(inhibitory effect upon cell death associated with diseases), cellsurvival maintaining effect, prophylactic and/or therapeutic activity(effect) on neurodegenerative diseases, cancers, immunological diseases,infections, gastrointestinal diseases, circulatory diseases andendocrine diseases] and yet encodes a polypeptide having a nature ofsubstantially the same quality as that of the polypeptide of theinvention. Specific examples of the polynucleotide encoding thepolypeptide of the invention include, but are not limited to, DNAscomprising a DNA having the nucleotide sequence as shown in SEQ ID NO:3.

DNAs which are capable of hybridizing to the nucleotide sequence asshown in SEQ ID NO: 3 under high stringency conditions may also beenumerated as the DNA encoding the polypeptide of the invention. Forexample, DNAs comprising a nucleotide sequence having about 80% or more,preferably about 85% or more, still more preferably about 90% or morehomology to the nucleotide sequence as shown in SEQ ID NO: 3 may beused.

Hybridization can be carried out according to known methods or methodsbased thereon, e.g. those methods described in “Molecular Cloning,” 2ndEd. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Whencommercial libraries are used, hybridization can be carried out inaccordance with the methods described in the attached instructions; morepreferably, hybridization is carried out under high stringencyconditions.

“High stringency conditions” refers to, for example, conditions wheresodium concentration is about 19–40 mM, preferably about 19–20 mM, andtemperature is about 50–70° C., preferably about 60–65° C.

As a DNA encoding a polypeptide having the amino acid sequence as shownin SEQ ID NO: 4, a DNA having the nucleotide sequence as shown in SEQ IDNO: 3 may be used, for example. As a DNA encoding the partial peptide ofthe invention, any DNA encoding the partial peptide of the invention maybe used. Specific examples of such DNA include a DNA comprising thenucleotide sequence as shown in SEQ ID NO: 4.

The cloning method of a DNA encoding the full length of the polypeptideor the partial peptide of the invention can be performed either by PCRamplification from genomic DNA or cDNA using synthetic DNA primers eachhaving a partial nucleotide sequence of the polypeptide or partialpeptide of the invention, or by a method where a DNA fragment isselected by hybridizing DNA inserted into an appropriate vector (i.e.library) to a DNA probe labeled with a ratio isotope or enzyme, the DNAprobe being a DNA fragment or a synthetic DNA encoding a part or fulllength of the polypeptide or partial peptide of the invention. Thehybridization can be carried out, for example, according to the methoddescribed in “Molecular Cloning”, 2nd Edition (J. Sambrook et al., ColdSpring Harbor Lab. Press, 1989). When commercial libraries are used, thehybridization can be carried out according to the attached instructions.

Substitution of the nucleotide sequence of a DNA can be performed byknown methods such as ODA-LA PCR, the gapped duplex method, the Kunkelmethod and the like using PCR, known kits such as Mutan™-Super ExpressKm (Takara), Mutan™-K (Takara), etc.

The DNA encoding the cloned polypeptide of the invention may be used asit is or after digestion with restriction enzymes or addition oflinkers, depending on purposes. The DNA may have ATG at its 5′ end as atranslation initiation codon and TAA, TGA, or TAG at its 3′ end as atranslation termination codon. The translation initiation andtermination codons may also be added by using appropriate synthetic DNAadapters.

Expression vectors for the polypeptide or the partial peptide of theinvention can be prepared by, for example, (a) cutting out desired DNAfragment from a DNA encoding the polypeptide or the partial peptide ofthe invention and (b) ligating the DNA fragment to an appropriateexpression vector downstream of its promoter.

Examples of vectors useful in the invention include plasmids derivedfrom Escherichia coli (e.g. pBR322, pBR325, pUC12, and pUC13); plasmidsderived from Bacillus subtilis (e.g. pUB110, pTP5 and pC194); plasmidsderived from yeast (e.g. pSH19 and pSH15); bacteriophages such asλ-phage; animal viruses such as retrovirus, vaccinia virus, baculovirus;and other vectors such as pA1-11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo andso on.

Any promoter may be used in the invention as long as it is appropriatefor the host that will be used for expressing a gene of interest. Whenthe host is an animal cell, examples of promoters useful in theinvention include SRα promoter, SV40 promoter, LTR promoter, CMVpromoter, HSV-TK promoter and β-actin promoter.

Among these promoters, CMV (cytomegalovirus) promoter, SRα promoter orthe like is preferably used. When the host is an Escherichia bacterium,trp promoter, lac promoter, recA promoter, λP_(L) promoter, lpppromoter, T7 promoter or the like is preferably used. When the host is aBacillus bacterium, SPO1 promoter, SPO2 promoter, penP promoter or thelike is preferably used. When the host is a yeast, PHO5 promoter, PGKpromoter, GAP promoter, ADH promoter, or the like is preferably used.When the host is an insect cell, polyhedrin promoter, P10 promoter orthe like is preferably used.

The expression vectors may, if desired, further comprise enhancers,splicing signals, polyadenylation signals, selective markers, SV40replication origin (hereinafter, sometimes abbreviated to “SV40 ori”)and the like. Examples of selective markers useful in the inventioninclude dihydrofolate reductase (hereinafter, sometimes abbreviated to“dhfr”) gene [methotorexate (MTX) resistance], ampicillin resistancegene (hereinafter, sometimes abbreviated to “Amp”), neomycin resistancegene [hereinafter, sometimes abbreviated to “Neo^(r)”: Geneticinresistance] and the like. When dhfr gene-deficient Chinese hamster cellsare used in combination with dhfr gene as a selective marker,recombinant cells may be selected even in a thymidine-free medium.

Furthermore, a signal sequence appropriate for the host may be added, ifnecessary, to the N-terminal of the polypeptide of the invention. Whenthe host is an Escherichia bacterium, the utilizable signal sequencesmay include PhoA signal sequence, OmpA signal sequence or the like maybe added. When the host is a Bacillus bacterium, α-amylase signalsequence, subtilisin signal sequence, or the like may be added. When thehost is yeast, MFα signal sequence, SUC2 signal sequence or the like maybe added. When the host is an animal cell, insulin signal sequence,α-interferon signal sequence, antibody molecule signal sequence, or thelike may be used.

Using the thus constructed vector comprising a DNA encoding thepolypeptide or the partial peptide of the invention, transformants canbe prepared.

Examples of hosts useful for this purpose include bacteria belonging tothe genus Escherichia, bacteria belonging to the genus Bacillus, yeasts,insect cells, insects, and animal cells.

Specific examples of bacteria belonging to the genus Escherichia usefulin the invention include E. coli K12 DH1 [Proc. Natl. Acad. Sci. USA,Vol. 60, 160 (1968)], JM103 [Nucleic Acids Research, Vol. 9, 309(1981)], JA221 [Journal of Molecular Biology, Vol. 120, 517 (1978)]),HB101 [Journal of Molecular Biology, Vol, 41, 459 (1969)] and C600[Genetics, Vol. 39, 440 (1954)].

Specific examples of bacteria belonging to the genus Bacillus useful inthe invention include B. subtilis MI114 [Gene, Vol. 24, 255 (1983)] and207–21 [Journal of Biochemistry, Vol. 95, 87 (1984)].

Specific examples of yeasts useful in the invention includeSaccharomyces cerevisiae AH22, AH22R⁻, NA87-11A, DKD-5D and 20B-12,Schizosaccharomyces pombe NCYC1913 and NCYC2036, and Pichia pastorisKM71.

Specific examples of insect cells useful in the invention include, whenthe virus used is AcNPV, a cell line derived from larvae of Spodopterafrugiperda (Sf cells), MG1 cells derived from the midgut of Trichoplusiani, High Five™ cells derived from eggs of Trichoplusia ni, Mamestrabrassicae-derived cells and Estigmena acrea-derived cells. When thevirus used is BmNPV, insect cells such as a silkworm-derived cell line(Bombyx mori N cells; BmN cells) may be used. Specific examples of Sfcells useful in the invention include Sf9 cells (ATCC CRL 1711) and Sf21cells [both disclosed in Vaughn J. L. et al., In Vivo, 13, 213–217(1977)].

Specific examples of insects useful in the invention include larvae ofsilkworm (Maeda et al., Nature, 315, 592 (1985)).

Specific examples of al cells useful in the invention include simiancell COS-7, Vero cells, Chinese hamster cell CHO (hereinafter,abbreviated to “CHO cells”), sdhfr gene-deficient Chinese hamster cellCHO (hereinafter, abbreviated to “CHO(dhfr⁻) cells”), mouse L cells,mouse AtT-20 cells, mouse myeloma cells, rat GH3 cells, and human FLcells.

Transformation of bacteria belonging to the genus Escherichia can beperformed in accordance with methods disclosed, for example, in Proc.Natl. Acad. Sci. USA, Vol. 69, 2110 (1972) and Gene, Vol. 17, 107(1982).

Transformation of bacteria belonging to the genus Bacillus can beperformed in accordance with methods disclosed, for example, inMolecular & General Genetics, Vol. 168, 111 (1979).

Transformation of yeasts can be performed in accordance with methodsdisclosed, for example, in Methods in Enzymology, 194, 182–187(1991) andProc. Natl. Acad. Sci. USA, Vo. 75, 1929 (1978).

Transformation of insect cells or insects can be performed in accordancewith methods disclosed, for example, in Bio/Technology, 6, 47–55 (1988).

Transformation of animal cells can be performed by methods disclosed,for example, in Cell Engineering, Separate Vol. 8, New Cell EngineeringExperiment Protocol, 263–267 (1995) (Shujunsha Co.) and Virology, Vol.52, 456 (1973).

Thus, transformants transformed with the expression vector comprising aDNA encoding the polypeptide can be obtained.

As a medium to culture transformants obtained from Escherichia orBacillus bacteria as hosts, a liquid medium is appropriate. The mediummay contain carbon sources, nitrogen sources, minerals, and so on whichare necessary for the growth of the transformant. As carbon sources,glucose, dextrin, soluble starch, sucrose or the like may be enumerated.As nitrogen sources, organic or inorganic substances such as ammoniumsalts, nitrates, corn steep liquor, peptone, casein, meat extract, beancake, potato extract, or the like may be enumerated. As minerals,calcium chloride, sodium dihydrogen phosphate, magnesium chloride, orthe like may be enumerated. Further, yeast, vitamins, growth-promotingfactors, etc. may also be added to the medium. Preferable pH of themedium is about 5–8.

As a medium to culture Escherichia bacteria, M9 medium containingglucose and casamino acid [Miller, Journal of Experiments in MolecularGenetics, 431–433, Cold Spring Harbor Laboratory, New York, (1972)] ispreferable, for example. If necessary, drugs such as 3 β-indolyl acrylicacid can be added to the medium to improve the efficiency of thepromoter.

When the host is an Escherichia bacterium, the transformant is culturedusually at about 15–43° C. for about 3–24 hours. If necessary, aerationand stirring may be applied.

When the host is a Bacillus bacterium, the transformant is culturedusually at about 30–40° C. for about 6–24 hours. If necessary, aerationand stirring may also be applied.

As a medium to culture transformants obtained from yeasts as hosts, amedium such as Burkholder minimum medium [Bostian, K. L. et al., Proc.Natl. Acad. Sci. USA. Vol. 77, 4505 (1980)] or SD medium containing 0.5%casamino acid [Bitter, G. A. et al., Proc. Natl. Acad. Sci. USA, Vol.81, 5330 (1984)] may be used, for example. It is preferable that the pHof the medium be adjusted to about 5–8. The transformant is culturedusually at about 20–35° C. for about 24–72 hours. If necessary, aerationand stirring may be applied.

As a medium to culture transformants obtained from insect cells orinsects as hosts, Grace's Insect Medium [Grace, T.C.C., Nature, 195, 788(1962)] supplemented with additives such as inactivated 10% bovine serummay be used, for example. It is preferable that the pH of the medium beadjusted to about 6.2–6.4. The transformant is cultured usually at about27° C. for about 3–5 days. If necessary, aeration and stirring may beapplied.

As a medium to culture transformants obtained from animal cells ashosts, examples of useful media include MEM medium [Science, Vol. 122,501 (1952)], DMEM medium [Virology, Vol. 8, 396 (1959)], RPMI 1640medium [Journal of the American Medical Association, Vol. 199, 519(1967)] and 199 medium [Proceedings of the Society of the BiologicalMedicine, Vol. 73, 1 (1950)] each containing about 5–20% fetal calfserum. Preferable pH of the medium is from about 6 to about 8. Thetransformant is cultured usually at about 30–40° C. for about 15–60hours. If necessary, aeration and stirring may be applied.

Thus, it is possible to allow the transformant to produce thepolypeptide or the partial peptide of the invention within cells or cellmembranes, or preferably, out of cells.

Separation and purification of the polypeptide or the partial peptide ofthe invention from the resultant culture can be carried out, forexample, according to the methods described below.

For extraction of the polypeptide or the partial peptide of theinvention from cultured microorganisms or cells, the microorganism cellsare harvested by known methods after the cultivation, suspended in asuitable buffer, and disrupted by sonication or by lysozyme and/orfreezing and thawing, etc. Then, a crude extract of the polypeptideextract is obtained by centrifugation or filtration. The buffer maycontain a protein denaturing agent such as urea or guanidinehydrochloride, or a surfactant such as Triton X-100™. If the polypeptideis secreted into the culture broth, the supernatant is separated fromthe microorganisms or cells after completion of the cultivation andcollected by known methods.

Purification of the polypeptide or the partial peptide of the inventioncontained in the resultant culture supernatant or extract can beperformed by an appropriate combination of known methods for separationand purification. These known methods include methods utilizingsolubility (such as salting out or sedimentation with solvents), methodsmainly utilizing difference in molecular weight (such as dialysis,ultrafiltration, gel filtration and SDS-polyacrylamide gelelectrophoresis), methods utilizing difference in electric charge (suchas ion-exchange chromatography), methods utilizing specific affinity(such as affinity chromatography), methods utilizing difference in thehydrophobicity (such as reversed-phase high-performance liquidchromatography), and methods utilizing difference in isoelectric point(such as isoelectric electrophoresis).

When the thus obtained polypeptide or partial peptide of the inventionis a free form, it can be converted into the above-described salt byknown methods or methods based thereon. On the contrary, when theprotein of interest is obtained in a salt form, the salt can beconverted into a free form or another salt according to known methods ormethods based thereon.

The polypeptide or the partial peptide produced by the transformant canbe arbitrarily modified or a part thereof can be removed therefrom byusing an appropriate protein modification enzyme before or after thepurification. Examples of such enzymes include trypsin, chymotrypsin,arginyl endopeptidase, protein kiliase and glycosidase.

The presence of the thus produced polypeptide or partial peptide of theinvention can be measured by enzyme immunoassays, Western blot analysis,etc. using specific antibodies.

Alternatively, the presence of the polypeptide or the partial peptide ofthe invention can be measured by fusing any foreign peptide sequence(e.g. FLAG, HIS tag, myc tag, HA tag, or HSV tag) that can be an epitope(antibody recognition site) to the N-terminal, C-terminal or some othersite of the polypeptide as described earlier and then detectingchemiluminescence or the like using an antibody that recognizes theabove peptide sequence.

Antibodies to the polypeptide or the partial peptide of the invention(hereinafter, sometimes referred to as the “antibody of the invention”)may be either polyclonal antibodies (hereinafter, sometimes referred toas the “polyclonal antibody of the invention”) or monoclonal antibodies(hereinafter, sometimes referred to as the “monoclonal antibody of theinvention”) as long as they can recognize the polypeptide or the partialpeptide of the invention.

The antibody to the polypeptide or the partial peptide of the inventioncan be prepared using the polypeptide or the partial peptide of theinvention as antigen and according to known methods for antibody oranti-serum preparation.

[Preparation of Monoclonal Antibodies]

(a) Preparation of Monoclonal Antibody-Producing Cells

The polypeptide or the partial peptide of die invention is administeredto warm-blooded animals either alone or together with a carrier ordiluent to a site capable of producing antibodies upon theadministration. In order to enhance the ability to produce antibodies,complete Freund's adjuvants or incomplete Freund's adjuvants may also beadministered. The administration is usually carried out once in everytwo to six weeks and two to ten times in the total. Examples ofwarm-blooded animals useful in the invention include monkey, rabbit,dog, guinea pig, mouse, rat, sheep, goat and chicken. Among them, mouseor rat is used preferably.

In the preparation of monoclonal antibody-producing cells, individualswith detectable antibody titers are selected from warm-blooded animals(e.g. mice) immunized with antigen. Then, the spleen or lymph nodes arecollected from them two to five days after the final immunization, andantibody-producing cells contained therein are fused with myeloma cellsof a homologous or heterologous animal to thereby obtain monoclonalantibody-producing hybridomas. Measurement of antibody titers inantisera may be carried out, for example, by reacting a labeledpolypeptide (which will be described later) with the antiserum, followedby measuring the activity of the labeling agent bound to the antibody.The cell fusion may be carried out by a known method, for example, themethod of Koehler and Milstein (Nature, 256, 495, (1975)). Examples ofuseful fusion promoters include polyethylene glycol (PEG), Sendai virus,etc. Preferably, PEG is used.

Examples of myeloma cells useful in the invention include myeloma cellsof warm-blooded animals such as NS-1, P3U1, SP2/0, AP-1, etc.Preferably, P3U1 is used. A preferable ratio of the number ofantibody-producing cells used (spleen cells) to the number of myelomacells is from about 1:1 to about 20:1. When PEG (preferably, PEG 1000 toPEG 6000) is added at a concentration of about 10–80% and the resultantcell mixture is incubated at 20–40° C. (preferably, at 30–37° C.) forabout 1–10 minutes, an efficient cell fusion can be achieved.

Various methods may be used for screening for monoclonalantibody-producing hybridomas. For example, hybridoma culturesupernatant is added to a solid phase (e.g. microplate) on which thepolypeptide antigen has been adsorbed either directly or with a carrier.Then, a radioactively or enzymatically labeled anti-immunoglobulinantibody (anti-mouse immunoglobulin antibody is used when mouse cellsare used in the cell fusion) or protein A is added thereto to detectmonoclonal antibodies bound to the solid phase. Alternatively, a methodmay be used in which hybridoma culture supernatant is added to a solidphase on which an anti-immunoglobulin antibody or protein A has beenadsorbed; then, a radioactively or enzymatically labeled polypeptide isadded thereto to thereby detect monoclonal antibodies bound to the solidphase.

Selection of monoclonal antibodies may be carried out by known methodsor methods based on them. Usually, selection can be carried out in amedium for culturing animal cells supplemented with HAT (hypoxanthine,aminopterin and thymidine). As a medium for selection and culturing, anymedium may be used as long as hybridomas are capable of growing therein.Examples of useful media include RPMI 1640 medium containing about 1–20%(preferably about 10–20%) of fetal calf serum, GIT medium (Wako PureChemical Industries, Ltd.) containing about 1–20% of fetal calf serumand a serum-free medium for hybridoma cultivation (SFM-101; NissuiPharmaceutical Co.). The cultivation temperature is usually about 20–40°C., preferably about 37° C. The cultivation period is usually from fivedays to three weeks, preferably one to two weeks. The cultivation may becarried out usually under 5% carbon dioxide. The antibody titer ofhybridoma culture supernatant may be measured in the same manner as inthe above-mentioned measurement of the antibody titers in antisera.

(b) Purification of the Monoclonal Antibodies

Separation and purification of monoclonal antibodies may be carried outby conventional methods, such as methods for separating/purifyingimmunoglobulin [e.g. salting-out, alcohol precipitation, isoelectricprecipitation, electrophoresis, adsorption/desorption using ionexchangers (e.g. DEAE), ultracentrifugation, gel filtration, specificpurification methods in which only an antibody is collected by means ofan antigen-binding solid phase or active adsorbent such as protein A orprotein G, followed by dissociation of the bond].

[Preparation of Polyclonal Antibodies]

The polyclonal antibody of the invention can be produced by knownmethods or methods based on them. For example, an immunogen (antigenpolypeptide) per se or a complex of the immunogen and a carrier proteinis prepared. Then, using the immunogen or the complex, warm-bloodedanimals are immunized in the same manner as described for the productionof monoclonal antibodies. Fractions containing the antibody against thepolypeptide or the partial peptide of the invention are harvested fromthe immunized animals, followed by separation and purification of theantibody.

With respect to the immunogen-carrier protein conjugate for use in theimmunization of warm-blooded animals, the kind of carrier protein andthe mixing ratio of the carrier and the hapten are not particularlyrestricted as long as antibodies are produced efficiently against thehapten cross-linked to the carrier. For example, bovine serum albumin,bovine thyroglobulin, hemocyanin, or the like is coupled to the haptenat a weight ratio of about 0.1–20:1, preferably about 1–5:1.

A variety of condensing agents can be used for the coupling between thehapten and the carrier. For example, glutaraldehyde, carbodiimide,maleimide, or active ester reagents containing a thiol or dithiopyridylgroup may be used.

The condensation product is administered to a warm-blooded animal eitheralone or together with a carrier or diluent at a site capable ofproducing antibodies upon the administration. In order to enhance theantibody production ability, complete Freund's adjuvant or incompleteFreund's adjuvant may also be administered. Administration is carriedout generally once in about every 2–6 weeks and about 3–10 times in thetotal.

Polyclonal antibodies can be recovered from the blood, abdominal dropsyor other body fluid, preferably from the blood, of the warm-bloodedanimal immunized as described above.

Polyclonal antibody titers in antisera can be determined in the samemanner as described above for the determination of monoclonal antibodytiters in antisera. The separation and purification of polyclonalantibodies can be carried by the same methods for separation andpurification of immunoglobulin as those described for the separation andpurification of monoclonal antibodies.

With respect to the antisense polynucleotide having a nucleotidesequence complementary to or substantially complementary to thepolynucleotide of the invention, any antisense polynucleotide may beused as long as it has a nucleotide sequence complementary to orsubstantially complementary to the polynucleotide of the invention andhas an effect capable of inhibiting the expression of the polynucleotide(DNA).

A nucleotide sequence substantially complementary to the polynucleotideof the invention refers to, for example, a nucleotide sequence havingabout 70% or more, preferably about 80% or more, more preferably about90% or more, most preferably about 95% or more homology to thefull-length or a partial nucleotide sequence of the complementarynucleotide sequence to the polynucleotide of the invention (i.e., thecomplementary strand to the DNA of the invention). Particularlypreferable is an antisense polynucleotide having about 70% or more,preferably about 80% or more, more preferably about 90% or more, mostpreferably about 95% or more homology to a part of the complementarystrand to the polynucleotide of the invention encoding an N-terminalportion of the polypeptide of the invention (e.g. nucleotide sequenceencoding a region neighboring the initiation codon). These antisensepolynucleotides can be synthesized with known DNA synthesizers.

When the polypeptide of the invention has a signal peptide, the peptideis efficiently secreted out of cells and manifests as a humoral factorimportant biological activities for signal transduction, self-defense,etc.

Hereinbelow, uses of the polypeptide of the invention, the partialpeptide of the invention (sometimes, these two and salts thereof arecollectively referred to as the “polypeptide of the invention”), thepolynucleotide encoding the polypeptide of the invention (thepolynucleotide of the invention), the antibody to the polypeptide of theinvention (the antibody of the invention) and the antisensepolynucleotide of the invention will be described.

(1) Therapeutic and/or Prophylactic Agents for Various Diseases wherethe Polypeptide of The Invention is Involved

The polypeptide of the invention exists in vivo, and has cell deathinhibitory effect, cell survival maintaining effect, etc. When thepolypeptide or the polynucleotide (e.g. DNA) of the invention ismutative, deficient, or expressed at an mutatively decreased or enhancedlevel, various diseases including diseases accompanied byneurodegeneration, such as neurodegenerative diseases [e.g. Alzheimer'sdisease (familial Alzheimer's disease, juvenile Alzheimer's disease,sporadic Alzheimer's disease, etc.), Parkinson's disease, Down syndrome,amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jacob disease,Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.],brain dysfuncytions (e.g. brain infarction, cerebral hemorrhage,subarachnoid hemorrhage, ischemic brain diseases, epidural hematoma,subdural hematoma, etc.), cancers (e.g. astrocytoma, oligodendroglioma,etc.), immunological diseases, infections (e.g. meningitis,protozoiasis, rickettsial infections, metazoan infections, bacterial orviral meningitis such as Borna's disease, postvaccinal encephalitis,AIDS encephalopathy, etc.), gastrointestinal diseases, circulatorydiseases and endocrine diseases develop.

Therefore, the polypeptide or the polynucleotide of the invention can beused as a medicine of low toxicity and high safety, for example, as acell death inhibitor and as a prophylactic and/or therapeutic agent forvarious diseases including diseases accompanied by neurodegeneration,such as neurodegenerative diseases [e.g. Alzheimer's disease (familialAlzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer's,disease, etc.), Parkinson's disease, Down syndrome, amyotropluc lateralsclerosis, prion disease, Creutzfeldt-Jacob disease, Huntington'schorea, diabetic neuropathy, multiple sclerosis, etc.], braindysfunctions (e.g. brain infarction, cerebral hemorrhage, subarachnoidhemorrhage, ischemic brain diseases, epidural hematoma, subduralhematoma, etc.), cancers (e.g. astrocytoma, oligodendroglioma, etc.),immunological diseases, infections (e.g. meningitis, protozoiasis,rickettsial infections, metazoan infections, bacterial or viralmeningitis such as Borna's disease, postvaccinal encephalitis, AIDSencephalopathy, etc.), gastrointestinal diseases, circulatory diseasesand endocrine diseases. Still preferably, the polypeptide or thepolynucleotide of the invention can be used as a prophylactic and/ortherapeutic agent for Alzheimer's disease.

For example, when a patient is suffering from insufficient or mutativesignal transduction resulted from decrease or deficiency of thepolypeptide of the invention in his/her body, it is possible to restoresufficient or normal function of the polypeptide of the invention by (1)administering the polynucleotide of the invention to the patient andthereby allowing the polypeptide of the invention to be expressed in thebody; (2) introducing the polynucleotide of the invention into cells tothereby allow the expression of the polypeptide of the invention, andthen transplanting the cells into the patient; or (3) administering thepolypeptide of the invention to the patient.

When the polynucleotide of the invention is used as the above-mentionedmedicine, the polynucleotide (e.g. DNA) per se or the polynucleotideinserted into an appropriate vector such as a retrovirus vector,adenovirus vector, adeno-associated virus vector, etc. may beadministered to human or other warm-blooded animals using conventionalmeans. The polynucleotide of the invention may be administered as it isor after formulation with physiologically acceptable carriers such asadjuvants to promote its uptake, by means of a gene gun or a cathetersuch as hydrogel catheter.

When the polypeptide of the invention is used as the above-describedprophylactic and/or therapeutic agent, at least 90%, preferably 95% ormore, more preferably 98% or more, still preferably 99% or more purifiedpolypeptide of the invention is used.

The polypeptide of the invention may be used, for example, orally in theform of tablets (sugar-coated, if necessary), capsules, elixirs,microcapsules or the like; or parenterally in the form of injectionssuch as aseptic solutions or suspensions in water or otherpharmaceutically acceptable liquids. These preparations may be produced,for example, by mixing the polypeptide of the invention withphysiologically acceptable carriers, flavoring agents, excipients,vehicles, antiseptics, stabilizers, binders, etc. in unit dosage formsrequired for preparing generally approved pharmaceutical preparations.The amounts of active ingredients in these formulations are decided sothat an appropriate dose within the specified range can be obtained.

Examples of additives which may be mixed in tablets, capsules, etc.include binders such as gelatin, corn starch, tragacanth and gum arabic,excipients such as crystalline cellulose, swelling agents such as cornstarch, gelatin and alginic acid, lubricants such as magnesium stearate,sweetening agents such as sucrose, lactose and saccharin, and flavoringagents such as peppermint, akamono oil and cherry. When the unit dosageform is capsule, liquid carrier such as oils and fats may further beincluded in addition to the above-mentioned materials. Sterilecompositions for injection can be formulated according to conventionalpractices in pharmaceutical manufacturing, e.g., by dissolving orsuspending active ingredients, naturally occurring vegetable oils suchas sesame oil, coconut oil, etc. in vehicles such as water forinjection.

Examples of aqueous liquids for injection include physiological salineand isotonic solutions containing glucose and other auxiliary agents(e.g. D-sorbitol, D-mannitol, sodium chloride, etc.). They may be usedin combination with a suitable auxiliary solubilizer such as alcohol(e.g. ethanol, etc.), polyalcohol (e.g. propylene glycol, polyethyleneglycol, etc.), nonionic surfactant (e.g. Polysorbate 80™, HCO-50, etc.).Examples of oily liquids for injection include sesame oil, soybean oil,etc. They may be used in combination with an auxiliary solubilizer suchas benzyl benzoate, benzyl alcohol, etc. In addition, buffers (e.g.phosphate buffer, sodium acetate buffer, etc.), analgesic agents (e.g.benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g.human serum albumin, polyethylene glycol, etc.), preservatives (e.g.benzyl alcohol, phenol, etc.), antioxidants, etc. may also be admixedtherewith. Usually, the prepared injections are filled in appropriateampoules.

Vectors into which the polynucleotide of the invention has beenintroduced may also be formulated as described above and usually usedparenterally.

Thus obtained preparations are safe and of low toxicity and they can beadministered to mammals (e.g., human, rat, mouse, guinea pig, rabbit,sheep, pig, bovine, horse, cat, dog, monkey, etc.).

Dose levels of the polypeptide of the invention may vary depending uponthe target disease, the patient to be treated, administration route, andso on. When the polypeptide of the invention is administered orally fortreating Alzheimer's disease, generally the polypeptide of the inventionis administered to adult patients (60 kg in body weight) at a dose ofabout 1–1000 mg/day, preferably about 10–500 mg/day, more preferablyabout 10–200 mg/day. With respect to parenteral administration, forexample, when the polypeptide of the invention is administered to adultpatients (60 kg in body weight) in the form of an injection for treatinga neurodegenerative disease such as Alzheimer's disease, it isconvenient to inject the polypeptide of the invention into the affectedpart of the body at a dose of about 1–1000 mg/day, preferably about1–200 mg/day, and more preferably about 10–100 mg/day, though the doseper administration may vary depending on the patient to be treated, thetarget disease, etc. For other animals, corresponding doses may beadministered after conversion of the above-mentioned values per 60 kgbased on actual body weights.

(2) Screening for Candidate Compounds for Medicine to Treat Diseases

Since the polypeptide of the invention exists in vivo, a compounds, or asalt thereof, that promotes the function of the polypeptide of theinvention may be used as a medicine of low toxicity and high safety, forexample, as a cell death inhibitor and as a prophylactic and/ortherapeutic agent for various diseases including diseases accompanied byneurodegeneration, such as neurodegenerative diseases [e.g. Alzheimer'sdisease (familial Alzheimer's disease, juvenile Alzheimer's disease,sporadic Alzheimer's disease, etc.), Parkinson's disease, Down syndrome,amyotroplic lateral sclerosis, prion disease, Creutzfeldt-Jacob disease,Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.],brain dysfunctions (e.g. brain infarction, cerebral hemorrhage,subarachnoid hemorrhage, ischemic brain diseases, epidural hematoma,subdural hematoma, etc.), cancers (e.g. astrocytoma, oligodendroglioma,etc.), immunological diseases, infections (e.g. meningitis,protozoiasis, rickettsial infections, metazoan infections, bacterial orviral meningitis such as Borna's disease, postvaccinal encephalitis,AIDS encephalopathy, etc.), gastrointestinal diseases, circulatorydiseases and endocrine diseases. Still preferably, the compound or asalt thereof can be used as a prophylactic and/or therapeutic agent forAlzheimer's disease.

On the other hand, a compounds, or a salt thereof, that inhibits thefunction of the polypeptide of the invention may be used as a medicinessuch as a prophylactic and/or therapeutic agent for diseases resultedfrom excessive production of the polypeptide of the invention (e.g.cancers).

Thus, the polypeptide of the invention is useful as a reagent forscreening for compounds, or salts thereof, that promote or inhibit thefunction of the polypeptide of the invention.

The present invention provides (1) a method for screening for compounds,or salts thereof, that promote or inhibit the activity (function) of thepolypeptide of the invention (hereinafter, sometimes just referred to asthe “promoter(s)” or “inhibitor(s)”), the method being characterized byusing the polypeptide of the invention.

Specifically, for example:

(2) a method of screening for the promoters or inhibitors is provided,wherein cell death inhibitory activities are compared between (i) whencells are contacted with the polypeptide of the invention and (ii) whencells are contacted with the polypeptide of the invention and a testcompound.

More specifically, in the above-described screening method, cells arecultured under the conditions of (i) and (ii), and then survival ratiosare measured.

As the cells, those cells in which cell death may be induced are usedpreferably. Specific example of cells useful in the invention include,but are not limited to, rat adrenal medulla-derived pheochromocytomacells (e.g. PC12h cells in Examples described later); rat or mousenerve-derived cell lines transformed with a vector comprising a DNAencoding the causative gene for familial Alzheimer's disease; andprimary culture of mouse cerebral cortex cells. The death of these cellsis induced by addition of glutamic acid, removal of serum, addition of βamyloid protein, or expression of the integrated DNA encoding thecausative gene of familial Alzheimer's disease.

The medium may be any medium as long as it does not inhibit the celldeath inhibitory effect of the polypeptide of the invention. Forexample, Dulbecco's modified Eagle's medium (DMEM) may be used.

Survival ratios may be measured by known methods, e.g., a method inwhich the lactate dehydrogenase (LDH) activity in cell extract ismeasured; MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromide) assay; MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay; trypan blue staining; or Calcein staining (Proc. Natl. Acad. Sci.USA 98: 6336–6341, 2001; NeuroReport 13:903–907, 2002; WO 01/21787).

The test compound may be, for example, a peptide, protein, non-peptidiccompound, synthetic compound, fermentation product, cell extract, plantextract, or animal tissue extract. These compounds may be either novelcompounds or known compounds.

For example, a test compound which promotes the cell death inhibitoryactivity (e.g. survival ratio) in (ii) above by about 20% or more,preferably by about 30% or more, more preferably by about 50% or morecompared to the activity in (i) above may be selected as a compound, ora salt thereof, that promotes the activity of the polypeptide of theinvention.

For example, a test compound which inhibits the cell death inhibitoryactivity (e.g. survival ratio) in (ii) above by about 20% or more,preferably by about 30% or more, more preferably by about 50% or morecompared to the activity in (i) above may be selected as a compound, ora salt thereof, that inhibits the activity of the polypeptide of theinvention.

The polynucleotide of the invention is useful as a reagent for screeningfor compounds, or salts thereof, that promote or inhibit the expressionof the polypeptide gene of the invention.

The present invention provides (3) a method of screening for compounds,or salts thereof, that promote or inhibit the expression of thepolypeptide gene of the invention (hereinafter, sometimes just referredto as the “promoter(s)” or “inhibitor(s)”), the method beingcharacterized by using the polypeptide of the invention. Morespecifically, for example:

(4) a method of screening for the promoters or inhibitors is provided,wherein the two cases of (iii) a cell capable of producing thepolypeptide of the invention is cultured and (iv) a mixture of the cellcapable of producing the polypeptide of the invention and a testcompound is cultured are compared.

In the above-described screening method, for example, the expressionlevels of the polypeptide gene of the invention (e.g. enzyme activitiesof alkali phosphatase, luciferase, etc. inserted downstream of thepromoter for the polypeptide gene of the invention; or levels of mRNAencoding the polypeptide of the invention) are measured and compared.

Specific examples of cells capable of producing the polypeptide of theinvention include, but not limited to, hosts (transformants) transformedwith a vector comprising a DNA encoding the polypeptide of theinvention. As the host, animal cells such as CHO cells may be usedpreferably. For the screening, those transformants are used preferablywhich produce the polypeptide of the invention within cells or in theculture supernatant when cultured as described above. More preferably,those transformants are used in which a gene encoding secretory alkaliphosphatase, luciferase or the like is inserted downstream of thepromoter for the polypeptide gene of the invention.

The test compound may be, for example, a peptide, protein, non-peptidiccompound, synthetic compound, fermentation product, cell extract, plantextract, or animal tissue extract. These compounds may be either novelcompounds or known compounds.

In order to practice the above-described screening method, cells capableof producing the polypeptide of the invention are prepared by culturingin a medium suitable for screening.

The medium may be any medium as long as it does not inhibit the celldeath inhibitory effect of the polypeptide of the invention. Forexample, DMEM may be used.

Expression levels of the polypeptide gene of the invention may bedetermined by measuring the enzyme activity of alkali phosphatase,luciferase or the like inserted downstream of the promoter for thepolypeptide gene of the invention, by conventional methods.

Alternatively, expression levels of the polypeptide gene of theinvention may also be measured by known methods such as Northernblotting, reverse transcription-polymerase chain reaction (RT-PCR), realtime PCR analysis system (ABI; TaqMan polymerase chain reaction), ormethods based thereon.

For example, a test compound which promotes the expression of thepolypeptide gene of the invention in (iv) above by about 20% or more,preferably by about 30% or more, more preferably by about 50% or morecompared to the expression in (iii) above may be selected as a compound,or a salt thereof, that promotes the expression of the polypeptide geneof the invention.

For example, a test compound which inhibits the expression of thepolypeptide gene of the invention in (iv) above by about 20% or more,preferably by about 30% or more, more preferably by about 50% or morecompared to the expression in (iii) above may be selected as a compound,or a salt thereof, that inhibits the expression of the polypeptide geneof the invention.

The polynucleotide of the invention is also useful as a reagent forscreening for compounds, or salts thereof, that promote or inhibit theactivity of the polypeptide gene of the invention.

The present invention provides (5) a method of screening for compounds,or salts thereof, that promote or inhibit the activity of thepolypeptide of the invention (hereinafter, sometimes just referred to asthe “promoter(s)” or “inhibitor(s)”), the method being characterized byusing the polynucleotide of the invention.

Specifically, for example:

(6) a method of screening for promoters or inhibitors of the cell deathinhibitory activity of the polypeptide of the invention is provided,wherein the two cases of (v) a cell capable of producing the polypeptideof the invention is cultured in contact with another cell and (vi) amixture of the cell capable of producing the polypeptide of theinvention and a test compound is cultured in contact with another cellare compared to thereby compare the activity of the polypeptide toinhibit the death of the another cell.

In the above screening methods, the cells are cultured under theconditions of (v) and (vi) above, and then survival ratios thereof aremeasured.

As the cell capable of producing the polypeptide of the invention, thecells mentioned in (4) above may be used. As the another cell, the cellsmentioned in (2) above may be used. The test compound, culturing method,method for measuring cell death inhibitory activities are as describedin (2) above.

For example, a test compound which promotes the cell death inhibitoryactivity (e.g. survival ratio) in (vi) above by about 20% or more,preferably by about 30% or more, more preferably by about 50% or morecompared to the activity in (v) above may be selected as a compound, ora salt thereof, that promotes the activity of the polypeptide of theinvention.

For example, a test compound which inhibits the cell death inhibitoryactivity (e.g. survival ratio) in (vi) above by about 20% or more,preferably by about 30% or more, more preferably by about 50% or morecompared to the activity in (v) above may be selected as a compound, ora salt thereof, that inhibits the activity of the polypeptide of theinvention.

The present invention provides (7) a method of screening for compounds,or salts thereof, that promote or inhibit the expression (production) ofthe polypeptide of the invention (hereinafter, sometimes just referredto as the “promoter(s)” or “inhibitor(s)”), the method beingcharacterized by using the antibody of the invention.

Specifically, for example:

(8) a method of screening for promoters or inhibitors is provided,wherein the two cases of (vii) a cell capable of producing thepolypeptide of the invention is cultured and (viii) a mixture of thecell capable of producing the polypeptide of the invention and a testcompound is cultured are compared using the antibody of the invention.

In the above-described screening method, for example, the yields of thepolypeptide of the invention in (vii) and (viii) are measured andcompared using the antibody of the invention.

The test compound may be, for example, a peptide, protein, non-peptidiccompound, synthetic compound, fermentation product, cell extract, plantextract, or animal tissue extract. These compounds may be either novelcompounds or known compounds.

In order to practice the above-described screening method, cells capableof producing the polypeptide of the invention are cultured in a mediumsuitable for screening. Any medium may be used as long as it does notinhibit the production of the polypeptide of the invention. For example,DMEM may be used.

Specific examples of cells capable of producing the polypeptide of theinvention include, but not limited to, hosts (transformants) transformedwith a vector comprising a DNA encoding the polypeptide of theinvention. As the host, animal cells such as CHO cells may be usedpreferably. For the screening, those transformants are used preferablywhich produce the polypeptide of the invention within cells or in theculture supernatant when cultured as described above.

The yields of the polypeptide of the invention may be measured byconventional methods, e.g. Western analysis of the polypeptide containedin cell extract using an antibody that recognizes the polypeptide,ELISA, or methods based thereon.

For example, a test compound which increases the yield of thepolypeptide of the invention (expression level) (viii) above by about20% or more, preferably by about 30% or more, more preferably by about50% or more compared to the yield in (vii) above may be selected as acompound, or a salt thereof, that promotes the expression of thepolypeptide of the invention.

For example, a test compound which decreases the yield of thepolypeptide of the invention (expression level) (viii) above by about20% or more, preferably by about 30% or more, more preferably by about50% or more compared to the yield in (vii) above may be selected as acompound, or a salt thereof that inhibits the expression of thepolypeptide of the invention.

The screening kit of the invention contains the polypeptide of theinvention or a salt thereof.

Compounds or salts thereof obtainable by using the screening method orscreening kit of the invention are compounds that are selected frompeptides, proteins, non-peptidic compounds, synthetic compounds,fermentation products, cell extracts, plant extracts, animal tissueextracts, plasma and so forth. They promote or inhibit the function ofthe polypeptide of the invention, the expression of the polypeptide geneof the invention, or the expression of the polypeptide of the invention.

As salts of such compounds, the same salts as described earlier on thesalts of the polypeptide of the invention may be used.

The compound or salt thereof that promotes the function of thepolypeptide of the invention; the compound or salt thereof that promotesthe expression of the polypeptide gene of the invention; or the compoundor salt thereof that promotes the expression of the polypeptide of theinvention each obtainable by the screening method or with the screeningkit of the invention, can be used as a medicine of low toxicity and highsafety, for example as a cell death inhibitor, and as a prophylacticand/or therapeutic agent for various diseases including diseasesaccompanied by neurodegeneration, such as neurodegenerative diseases[e.g. Alzheimer's disease (familial Alzheimer's disease, juvenileAlzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson'sdisease, Down syndrome, amyotrophic lateral sclerosis, prion disease,Creutzfeldt-Jacob disease, Huntington's chorea, diabetic neuropathy,multiple sclerosis, etc.], brain dysfunctions (e.g. brain infarction,cerebral hemorrhage, subarachnoid hemorrhage, ischemic brain diseases,epidural hematoma, subdural hematoma, etc.), cancers (e.g. astrocytoma,oligodendroglioma, etc.), immunological diseases, infections (e.g.meningitis, protozoiasis, rickettsial infections, metazoan infections,bacterial or viral meningitis such as Borna's disease, postvaccinalencephalitis, AIDS encephalopathy, etc.), gastrointestinal diseases,circulatory diseases and endocrine diseases. Preferably, the compound ora salt thereof may be used as a prophylactic and/or therapeutic agentfor neurodegenerative diseases and brain dysfunctions. More preferably,the compound or a salt thereof may be used as a prophylactic and/ortherapeutic agent for Alzheimer's disease.

On the other hand, the compound or salt thereof that inhibits thefunction of the polypeptide of the invention; the compound or saltthereof that inhibits the expression of the polypeptide gene of theinvention; or the compound or salt thereof that inhibits the expressionof the polypeptide of the invention can be used as a medicine, forexample, as a prophylactic and/or therapeutic agent for diseasesresulted from excessive production of the polypeptide of the invention(e.g. cancers).

When a compound obtainable by using the screening method or screeningkit of the invention is used as the above-described therapeutic and/orprophylactic agent, the compound may be used by conventional means. Forexample, the compound may be formulated into tablets, capsules, elixirs,microcapsules, aseptic solutions, suspensions, etc. in the same manneras described for the medicine comprising the polypeptide of theinvention.

Thus obtained preparations are safe and of low toxicity, so they can beadministered to mammals (e.g. human, rat, mouse, guinea pig, rabbit,sheep, pig, bovine, horse, cat, dog, monkey, etc.).

Dose levels of these compounds or salts thereof may vary depending upontheir effect, the target disease, the patient to be treated,administration route, and so on. For example, when a compound thatpromotes the activity or function of the polypeptide of the invention isadministered orally for treating Alzheimer's disease, generally thecompound is administered to adult patients (60 kg in body weight) at adose of about 0.1–100 mg/day, preferably about 1.0–50 mg/day, morepreferably about 1.0–20 mg/day. With respect to parenteraladministration, when a compound that promotes the activity or functionof the polypeptide of the invention is administered to adult patients(60 kg in body weight) in the form of an injection for treatingAlzheimer's disease, it is convenient to intravenously inject thecompound at a dose of about 0.01–30 mg/day, preferably about 0.1–20mg/day, and more preferably about 0.1–10 mg/day, though the dose peradministration may vary depending on the patient to be treated, thetarget disease, etc. For other animals, corresponding doses may beadministered after conversion of the above-mentioned values per 60 kgbased on actual body weights.

On the other hand, when a compound that inhibits the activity orfunction of the polypeptide of the invention is administered orally,generally the compound is administered to adult patients (60 kg in bodyweight) at a dose of about 0.1–100 mg/day, preferably about 1.0–50mg/day, more preferably about 1.0–20 mg/day. With respect to parenteraladministration, a compound that inhibits the function of the polypeptideof the invention is administered to adult patients (60 kg in bodyweight) in the form of an injection, it is convenient to intravenouslyinject the compound at a dose of about 0.01–30 mg/day, preferably about0.1–20 mg/day, and more preferably about 0.1–10 mg/day, though the doseper administration may vary depending on the patient to be treated, thetarget disease, etc. For other animals, corresponding doses may beadministered after conversion of the above-mentioned values per 60 kgbased on actual body weights.

(3) Quantitative Determination of the Polypeptide Using the Antibody ofthe Invention

Since the antibody of the invention can specifically recognize thepolypeptide of the invention, the antibody may be used for quantitativedetermination of the polypeptide of the invention contained in samplesolutions, in particular, in quantitative determination by sandwichimmunoassay.

The present invention provides:

-   (i) a method of quantitative determination of the polypeptide of the    invention in a sample solution, comprising competitively reacting    the antibody of the invention with the sample solution and the    polypeptide of the invention labeled, and determining the ratio of    the, labeled polypeptide of the invention bound to the antibody; and-   (ii) a method of quantitative determination of the polypeptide of    the invention in a sample solution, comprising reacting the sample    solution with the antibody of the invention insolubilized on a    carrier and another antibody of the invention labeled,    simultaneously or in succession and determining the activity of the    labeling agent on the insolubilized carrier.

Further, the monoclonal antibody of the invention may be used toquantitatively determine the polypeptide of the invention or may be usedfor detection of the polypeptide by tissue staining. For these purposes,either antibody molecules per se or the F(ab′)₂, Fab′ or Fab fragmentthereof may be used.

Methods of quantitative determination of the polypeptide of theinvention using the antibody of the invention are not particularlylimited. Any measuring method may be used in which the amount ofantibody, antigen or antibody-antigen complex corresponding to theamount of the antigen in a sample solution (e.g. the amount of thepolypeptide of the invention) is detected by chemical or physical means,and then calculated from a standard curve prepared with a standardsolution containing a known amount of the antigen. For example,nephrometry, competitive methods, immunometric methods and sandwichassay may be used conveniently and, in terms of sensitivity andspecificity, the sandwich assay described later is particularlypreferred.

Examples of labeling agents useful in measuring methods utilizinglabeling substances include radioisotopes, enzymes, fluorescentsubstances, and luminescent substances. Examples of radioisotopesinclude [¹²⁵I], [¹³¹I], [³H] and [¹⁴C]. Preferred examples of enzymesare those which are stable and with high specific activity, e.g.,β-galactosidase, β-glucosidase, alkali phosphatase, peroxidase andmalate dehydrogenase. Examples of fluorescent substances includefluorescamine and fluorescein isothiocyanate. Examples of luminescentsubstances include luminol, luminol derivatives, luciferin, andlucigenin. Further, a biotin-avidin system may also be used for bindingan antibody or antigen with a labeling agent.

Insolubilization of antigens or antibodies may be performed by physicaladsorption or by chemical binding usually used for insolubilizing orimmobilizing polypeptides or enzymes. Examples of carriers useful forthis purpose include insoluble polysaccharides such as agarose, dextranand cellulose; synthetic resins such as polystyrene, polyacrylamide andsilicone; and glass.

In the sandwich assay, a sample solution is reacted with aninsolubilized monoclonal antibody of the invention (primary reaction);then, another monoclonal antibody of the invention that is labeled isreacted therewith (secondary reaction); and the activity of the labelingagent on the insolubilized carrier is measured to thereby quantitativelydetermine the amount of the polypeptide of the invention in the samplesolution. The primary reaction and the secondary reaction may beconducted in the reverse order, or they may be conducted simultaneouslyor with an interval. The type of the labeling agent and the method ofinsolubilization may be the same as those described herein earlier. Inimmunoassays using the sandwich technique, the antibody insolubilized ona solid phase or the antibody labeled is not necessarily a singleantibody; a mixture of two or more antibodies may be used for thepurposes of enhancing the sensitivity of measurement, etc.

In the method of measuring the polypeptide of the invention by thesandwich assay of the invention, the monoclonal antibodies of theinvention used in the primary and the secondary reactions are preferablythose antibodies whose sites of binding to the polypeptide of theinvention are different from each other. For example, if the antibodyused in the secondary reaction recognizes the C-terminal region of thepolypeptide of the invention, an antibody that recognizes a site otherthan the C-terminal region, e.g. an N-terminal region, is preferablyused in the primary reaction.

The monoclonal antibody of the invention may be used in a measuringsystem other than the sandwich assay, such as competitive methods,immunometric methods and nephrometry.

Incompetitive methods, an antigen in a sample solution and a labeledantigen are reacted competitively with an antibody; then, unreactedlabeled antigen (F) and labeled antigen bound to the antibody (B) areseparated (i.e. B/F separation); and the amount of the label of B or Fis measured to thereby quantitatively determine the amount of theantigen in the sample solution. With respect to this reaction method,there are a liquid phase method in which a soluble antibody is used; andthe B/F separation is conducted with polyethylene glycol, and a secondantibody to the above-mentioned antibody is used; and a solid phasemethod in which a solidified antibody is used as the first antibody or asoluble antibody is used as the first antibody while a solidifiedantibody is used as the second antibody.

In immunometric methods, an antigen in a sample solution and asolidified antigen are reacted competitively with a specific amount of alabeled antibody, followed by separation of the solid phase from theliquid phase; or an antigen in a sample solution is reacted with anexcessive amount of a labeled antibody, and then a solidified antigen isadded to bind unreacted labeled antibody to the solid phase, followed byseparation of the solid phase from the liquid phase. Subsequently, theamount of label in one of the phases is measured to determine the amountof the antigen in the sample solution.

In nephrometry, the amount of insoluble precipitate generated as aresult of antigen-antibody reaction in a gel or solution is measured.Even when the amount of the antigen in a sample solution is small andthus only a small amount of such precipitate is obtained, lasernephrometry utilizing the scattering of laser can be used conveniently.

In applying each of those immunological measuring methods to themeasuring method of the present invention, no special conditions oroperations are required. A measuring system for the polypeptide of thepresent invention may be constructed using the conventional conditionsand operational procedures in the relevant measuring method while takinginto account usual technical consideration of those skilled in the art.For details of these commonly used technical means, a variety ofreviews, reference books, etc. may be referred to.

For example, Hiroshi Irie (ed.): “Radioimmunoassay” (Kodansha, 1974);Hiroshi Irie (ed.): “Radioimmunoassay; Second Series” (Kodansha, 1979);Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Igaku Shoin, Japan,1978); Eiji Ishikawa et al. (ed.): “Enzyme Immunoassay” (Second Edition)(Igaku Shoin, 1982); Eiji Ishikawa et al. (ed.): “Enzyme Immnunoassay”(Third Edition) (Igaku Shoin, 1987); “Methods in Enzymology”, Vol. 70(Immunochemical Techniques (Part A)); ibid., Vol. 73 (ImmunochemicalTechniques (Part B)); ibid., Vol. 74 (Immunochemical Techniques (PartC)); ibid., Vol. 84 (immunochemical Techniques (Part D: SelectedImmunoassays)); ibid., Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid., Vol. 121(Immunochemical Techniques (Part 1: Hybridoma Technology and MonoclonalAntibodies)) (Academic Press) and the like may be referred to.

By using the antibody of the invention as described above, thepolypeptide of the invention can be quantitatively determined with highsensitivity.

Further, when an increase or decrease is detected in the concentrationof the polypeptide of the invention in a subject by quantitativelydetermining the concentration of the polypeptide of the invention usingthe antibody of the invention, it is possible to diagnose that thesubject has one of the following diseases: for example, diseasesaccompanied by neurodegeneration, such as neurodegenerative diseases[e.g. Alzheimer's disease (familial Alzheimer's disease, juvenileAlzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson'sdisease, Down syndrome, amyotrophic lateral sclerosis, prion disease,Creutzfeldt-Jacob disease, Huntington's chorea, diabetic neuropathy,multiple sclerosis, etc.], brain dysfunctions (e.g. brain infarction,cerebral hemorrhage, subaraclinoid hemorrhage, ischemic brain diseases,epidural hematoma, subdural hematoma, etc.), cancers (e.g. astrocytoma,oligodendroglioma, etc.), immunological diseases, infections (e.g.meningitis, protozoiasis, rickettsial infections, metazoan infections,bacterial or viral meningitis such as Borna's disease, postvaccinalencephalitis, AIDS encephalopathy, etc.), gastrointestinal diseases,circulatory diseases and endocrine diseases; or that the subject is verylikely to develop such a disease in the future.

Further, the antibody of the invention may be used for detecting thepolypeptide of the invention present in body fluids, tissues or othersamples. The antibody of the invention may also be used in thepreparation of antibody columns for use in the purification of thepolypeptide of the invention; in the detection of the polypeptide of theinvention in individual fractions generated in the course ofpurification; and in the analysis of the behavior of the polypeptide ofthe invention in test cells.

(4) Diagnostic Agents Comprising the Polynucleotide of the Invention

The polynucleotide of the invention can, when used as a probe forexample, detect mutativeities in DNA or mRNA encoding the polypeptide ofthe invention (gene mutativeities) in mammals (e.g. human, rat, mouse,guinea pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).Thus, the polynucleotide of the invention is useful as a gene diagnosticfor diagnosing, e.g., damage, mutations or reduced expression of theabove DNA or mRNA, or increase or excessive expression of the above DNAor mRNA.

Gene diagnosis using the polynucleotide of the invention may beperformed by known methods such as Northern hybridization or PCR-SSCPmethod (Genomics, Vol. 5, 874–879 (1989); Proceedings of the NationalAcademy of Sciences of the USA, 86: 2766–2770 (1989)).

When a decrease in expression is detected by Northern hybridization orwhen a mutation(s) is/are detected in the DNA by PCR-SSCP method, forexample, it is possible to diagnose that the relevant subject is verylikely to have one of the following diseases: for example, diseasesaccompanied by neurodegeneration, such as neurodegenerative diseases[e.g. Alzheimer's disease (familial Alzheimer's disease, juvenileAlzheimer's disease, sporadic Alzheimer's disease, etc.), Parkinson'sdisease, Down syndrome, amyotrophic lateral sclerosis, prion disease,Creutzfeldt-Jacob disease, Huntington's chorea, diabetic neuropathy,multiple sclerosis, etc.], brain dysfunctions (e.g. brain infarction,cerebral hemorrhage, subarachnoid hemorrhage, ischemic brain diseases,epidural hematoma, subdural hematoma, etc.), cancers (e.g. astrocytoma,oligodendroglioma, etc.), immunological diseases, infections (e.g.meningitis, protozoiasis, rickettsial infections, metazoan infections,bacterial or viral meningitis such as Borna's disease, postvaccinalencephalitis, AIDS encephalopathy, etc.), gastrointestinal diseases,circulatory diseases and endocrine diseases.

(5) Medicines and Diagnostic Agents Containing Antisense Polynucleotide

Antisense polynucleotide that complementarily binds to thepolynucleotide of the invention and thus inhibits the expression of thatpolynucleotide can inhibit the function of the polypeptide or thepolynucleotide of the invention in vivo. Therefore, the antisensepolynucleotide may be used as prophylactic and/or therapeutic agents fordiseases resulted from excessive expression of the polypeptide of theinvention (e.g. cancers).

The above-mentioned antisense polynucleotide may be used as theabove-mentioned prophylactic and/or therapeutic agents in the samemanner as the various prophylactic and/or therapeutic agents containingthe polynucleotide of the invention described earlier.

For example, the antisense polynucleotide per se or the antisensepolynucleotide inserted into an appropriate vector such as a retrovirusvector, adenovirus vector, adeno-associated virus vector, etc. may beadministered using conventional means. The antisense polynucleotide maybe administered as it is or after formulation with physiologicallyacceptable carriers such as adjuvants to promote uptake, by means of agene gun or a catheter such as hydrogel catheter.

Further, the antisense polynucleotide may be used as an oligonucleotideprobe for diagnostic purposes to examine the presence or state ofexpression of the polynucleotide of the invention in tissues or cells.The antisense polynucleotide may be used for diagnosis of, for example,diseases accompanied by neurodegeneration, such as neurodegenerativediseases [e.g. Alzheimer's disease (familial Alzheimer's disease,juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.),Parkinson's disease, Down syndrome, amyotroplhic lateral sclerosis,prion disease, Creutzfeldt-Jacob disease, Huntington's chorea, diabeticneuropathy, multiple sclerosis, etc.], brain dysfunctions (e.g. braininfarction, cerebral hemorrhage, subaracloid hemorrhage, ischemic braindiseases, epidural hematoma, subdural hematoma, etc.), cancers (e.g.astrocytoma, oligodendroglioma, etc.), immunological diseases,infections (e.g. meningitis, protozoiasis, rickettsial infections,metazoan infections, bacterial or viral meningitis such as Borna'sdisease, postvaccinal encephalitis, AIDS encephalopathy, etc.),gastrointestinal diseases, circulatory diseases-and endocrine diseases.

(6) Medicines Containing the Antibody of the Invention

The antibody of the invention that has an effect of neutralizing theactivity of the polypeptide of the invention may be used as prophylacticand/or therapeutic agents for diseases resulted from excessiveexpression of the polypeptide of the invention (e.g. cancers).

The above-mentioned prophylactic and/or therapeutic agents comprisingthe antibody of the invention may be administered orally or parenterallyto mammals (e.g. human, rat, rabbit, sheep, pig, bovine, cat, dog,monkey, etc.) in the forms of liquid preparations without any processingor in appropriate forms of pharmaceutical compositions. Dose levels mayvary depending upon the patient to be treated, the target disease,symptoms, administration route, and so on. However, it is convenient toinject the antibody of the invention intravenously at a dose of about0.01–20 mg/kg body weight, preferably about 0.1–10 mg/kg body weight,more preferably about 0.1–5 mg/kg body weight per administration aboutone to five times a day, preferably about one to three times a day. Inother parenteral administration and oral administration, similar doselevels may be used. If symptoms are particularly heavy, the dose may beincreased accordingly.

The antibody of the invention may be administered per se or in the formsof appropriate pharmaceutical compositions. The pharmaceuticalcompositions for the above administration comprise the antibody or saltthereof, pharmacologically acceptable carriers, and diluents orexcipients. Such compositions are provided in forms appropriate for oralor parenteral administration.

For example, compositions for oral administration include solid orliquid preparations such as tablets (including sugar-coated tablets andfilm-coated tablets), pills, granules, dispersants, capsules (includingsoft capsules), syrups, emulsions and suspensions. These compositionsare prepared according to conventional methods and contain carriers,diluents or excipients conventionally used in the field of medicinemanufacturing. For example, lactose, starch, sucrose, magnesium stearateand the like are used as carriers or excipients for tablets.

Compositions for parenteral administration include, for example,injections and suppositories. Injections include intravenous injections,subcutaneous injections, intradermal injections, muscle injections,instilment injections, etc. Such injections may be prepared bydissolving, suspending or emulsifying the above antibody or salt thereofin an aseptic, aqueous or oily liquid. Examples of aqueous liquids forinjection include physiological saline and isotonic solutions containingglucose and other auxiliary agents. They may be used in combination witha suitable auxiliary solubilizer such as alcohol (e.g. ethanol),polyalcohol (e.g. propylene glycol, polyethylene glycol), nontionicsurfactant [e.g. Polysorbate 80™, HCO-50 (polyoxyethylene (50 mol)adduct of hydrogenated castor oil)], etc.). Examples of oily liquids forinjection include sesame oil and soybean oil. They may be used incombination with an auxiliary solubilizer such as benzyl benzoate,benzyl alcohol, etc. Usually, the prepared injections are filled inappropriate ampoules. Suppositories for administration into rectum maybe prepared by mixing the antibody or a salt thereof with a conventionalsuppository base.

It is convenient to formulate the above-described pharmaceuticalcompositions for oral or parenteral administration into unlit dosageforms that would give an appropriate dose of the active ingredient.Examples of such unit dosage forms include tablets, pills, capsules,injections (ampoules), and suppositories. Usually, each unit of thesedosage forms contains preferably about 5–500 mg of the above-describedantibody. In particular, each unit contains preferably about 5–100 mg ininjections, and each unit in other dosage forms contains preferablyabout 10–250 mg.

The above-described pharmaceutical compositions may contain other activeingredients as long as they do not produce undesirable interaction withthe above-described antibody.

(7) DNA-Transferred Animals

The present invention further provides non-human mammals harboring aforeign DNA coding for the polypeptide of the invention (hereinafterreferred to briefly as the “foreign DNA of the invention”) or a mutantthereof (sometimes referred to briefly as the “foreign mutant DNA of theinvention”).

Thus, the present invention provides:

-   (1) A non-human mammal harboring the foreign DNA of the invention or    a mutant DNA thereof:-   (2) The non-human mammal according to (1) which is a rodent:-   (3) The non-human mammal according to (2) wherein the rodent is    mouse or rat; and-   (4) A recombinant vector containing the foreign DNA of the invention    or a mutant DNA thereof and capable of expressing the DNA in a    mammal.

The non-human manual harboring the foreign DNA of the invention or amutant DNA thereof (hereinafter referred to briefly as the“DNA-transferred animal of the invention”) can be created bytransferring the DNA of interest to a germinal cell such as unfertilizedegg cells, fertilized egg cells, or sperm cells or primordial cellsthereof, preferably during the period of embryogenesis in theontogenesis of the non-human mammal (more preferably, in the stage of asingle cell or a fertilized egg cell and generally at the 8-cell stageor earlier), by the calcium phosphate method, electric pulse method,lipofection method, agglutination method, microinjection method,particle gun method, or DEAE-dextran method. It is also possible totransfer the foreign DNA of the invention of interest into somaticcells, organs in the living body, tissue cells, or the like by such DNAtransfer methods to use the resultant cells or tissues in cell cultureor tissue culture. Further, by fusing the resultant cells with theabove-mentioned germinal cell by known cell fusion methods, it is alsopossible to create the DNA-transferred animal of the invention.

The non-human manual useful in the invention includes bovine, pig,sheep, goat, rabbit, dog, cat, guinea pig, hamster, mouse, rat, and soon. From the viewpoint of construction of diseased animal models,rodents which have comparatively short ontogenesis and life cycles andcan be easily bred, particularly mouse (e.g. pure strains such asC57BL/6, DBA2, etc. and hybrid strains such as B6C3F₁, BDF₁, B6D2F₁,BALB/c, ICR, etc.) or rat (e.g. Wistar, SD, etc.), are preferred.

As the “mammal” in the expression “a recombinant vector . . . capable ofexpressing the DNA in a mammal”, human may also be enumerated inaddition to the above-mentioned non-human mammals.

The foreign DNA of the invention is not a DNA of the invention which isinherently possessed by the non-human mammal, but a DNA of the inventionthat has been once isolated and extracted from a mammal.

Examples of the mutant DNAs of the invention include not oily the DNAsthat have variations (e.g. mutations) in the nucleotide sequence of theoriginal DNA of the invention as a result of, for example, addition ordeletion of nucleotides or substitution with other nucleotides, but alsomutative DNAs.

The term “mutative DNA” as used herein means any DNA that causesexpression of a mutative polypeptide of the invention. For example, aDNA that allows expression of a polypeptide that inhibits the functionof the normal polypeptide of the invention may be used.

The foreign DNA of the invention may be derived from a mammal that is ofthe same species as that of the host animal or of different species. Fortransferring of the DNA of the invention to the host animal, it isgenerally advantageous to use a DNA construct in which the DNA isligated downstream of a promoter capable of expressing the DNA in animalcells. For example, in transferring the human DNA of the invention, thishuman DNA of the invention may be ligated downstream of a promotercapable of directing expression of DNAs derived from various animals(e.g. rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.) harboringthe DNA of the invention having high homology to the human DNA tothereby prepare a DNA construct (e.g. vector), which can then bemicroinjected into fertilized egg cells of a host manual such asfertilized mouse egg cells. Thus, a DNA-transferred manual showing highexpression of the DNA of the invention can be created.

Examples of the expression vector for the polypeptide of the inventioninclude plasmids derived from E. coli, plasmids derived from B.subtilis, plasmids derived from yeast, λ phage and other bacteriophages,retroviruses such as Molony leukemia virus, and animal viruses such asvaccinia virus and vaculovirus. Preferable examples are E. coli-derivedplasmids, B. subtilis-derived plasmids and yeast-derived plasmids.

Examples of promoters that regulate the expression of the DNA include(1) promoters for DNAs derived from viruses (e.g. simian virus,cytomegalovirus, Molony leukemia virus, JC virus, papilloma virus,poliovirus, etc.), (2) promoters derived from mammals (e.g. human,rabbit, dog, cat, guinea pig, hamster, rat, mouse, etc.), for example,promoters of albumin, insulin II, uroprakin II, elastase,erythropoietin, endothelin, muscle creatine kinase, glial fibrillaryacidic protein, glutathione S-transferase, platelet-derived growthfactor β, keratin K1, K10, and K14, collagen type I and type II, cyclicAMP-dependent polypeptide kinase βI subunit, dystrophin, tartaricacid-resistant alkaline phosphatase, atrial natriuretic factor,endothelial receptor tyrosine kinase (generally abbreviated to Tie2),sodium/potassium-dependent adenosine triphosphatase (Na, K-ATPase),neurofilament light chain, metallothionein I and IIA, metalloproteiniaseI tissue inhibitor, MHC Class I antigen (H-2L), H-ras, renin, dopamineβ-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongationfactor 1α (EF-1α), β-actin, α- and β-myosin heavy chain, myosin lightchains 1 and 2, myelin basic protein, thyroglobulin, Thy-1,immunoglobulin H chain variable region (VNP), serum amyloid P component,myoglobin, troponin C, smooth muscle α-actin, preproenkephalin A,vasopressin, and so on. Preferable are those promoters which can directhigh expression of the DNA in the whole body, e.g. cytomegaloviruspromoter, human polypeptide chain elongation factor 1α (EF-1α) promoterand human and chicken β-actin promoters.

It is preferable that the vector has a sequence for terminating thetranscription of the mRNA of interest (generally called terminator) inthe DNA-transferred mammal. For example, sequences derived from virusesor various mammals may be used. Preferably, SV40 terminator derived fromsimian virus or the like is used.

In addition, for enhancing the expression of the DNA of interestfurther, it is possible, depending on the specific purpose, to ligate asplicing signal, an enhancer domain, a portion of an eucaryotic DNAintron, etc. upstream of the 5′-end of the promoter region, between thepromoter region and the translated region, or downstream of the 3′-endof the translated region.

The translated region can be prepared as a DNA construct which can beexpressed in a DNA-transferred animal, by conventional recombinant DNAtechniques, i.e. by ligating it downstream of the promoter and, ifdesired, upstream of the transcription termination site.

The transfer of the foreign DNA of the invention at the fertilized eggcell stage insures that the DNA will be ubiquitous in all the germ cellsand somatic cells of the host mammal. The presence of the DNA of theinvention in the germ cells of the DNA-transferred animal following DNAtransfer means that all the germinal cells and somatic cells of all thesubsequent generations of the DNA-transferred animal harbor the DNA ofthe invention. Thus, the progeny of such DNA-transferred animal whichinherited the foreign DNA of the invention have the DNA in all of theirgerm cells and somatic cells.

The non-human mammal harboring the foreign normal DNA of the inventioncan be verified by mating to retain the foreign DNA stably and then bredas a line harboring that DNA from generation to generation under usualbreeding conditions.

The transfer of the foreign DNA of the invention at the fertilized eggcell stage insures that the DNA will be present in excess in all thegerm cells and somatic cells of the host mammal. The presence of theforeign DNA of the invention in the germ cells of the DNA-transferredanimal following the DNA transfer means that all the germinal cells andsomatic cells of all the progeny of the DNA-transferred animal harborthe foreign DNA of the invention in excess. Thus, the progeny of suchDNA-transferred animal which inherited the foreign DNA of the inventionhave the DNA in excess in their germ cells and somatic cells.

By preparing homozygous animals having the transferred DNA in bothhomologous chromosomes and mating male animals with female animals, itis possible to breed through generations so that every progeny harborsthe DNA in excess.

The non-human mammal harboring the normal DNA of the invention featuresa high expression of the normal DNA and may eventually develop ahyperergasia of the polypeptide of the invention though activation ofthe function of the endogenous normal DNA. Thus, the animal can beutilized as an animal model of that disease. For example, by using theDNA-transferred animal harboring the normal DNA of the invention, it ispossible to study the hyperergasia of the polypeptide of the invention,to elucidate the mechanisms of diseases with which the polypeptide ofthe invention is associated, and to explore therapeutic modalities forthe diseases.

Furthermore, the mammal to which the foreign normal DNA of the inventionhas been transferred presents symptoms due to an increase in the freepolypeptide of the invention and, therefore, can also be used in thescreening of therapeutic drugs for diseases with which the polypeptideof the invention is associated.

On the other hand, the non-human mammal harboring the foreign mutativeDNA of the invention can be verified by mating to retain the DNA stablyand then bred as a line harboring the DNA from generation to generationunder usual breeding conditions. Moreover, it is possible to incorporatethe foreign DNA of interest in the above-mentioned plasmid and use it asa starting material. The DNA construct with the promoter can be preparedby conventional recombinant DNA techniques. Transfer of the mutative DNAof the invention in the fertilized egg cell stage insures that thetransferred DNA will be ubiquitous in all the germ cells and somaticcells of the host mammal. The presence of the mutative DNA of theinvention in the germ cells of the DNA-transferred animal means that allthe progeny of this DNA-transferred animal harbor the mutative DNA ofthe invention in all of their germinal cells and somatic cells. Theprogeny of this animal harbor the mutative DNA of the invention in allof their germinal cells and somatic cells. By preparing homozygous maleand female animals having the introduced DNA in both homologouschromosomes and mating them, it can be insured that every progenyharbors the DNA from generation to generation.

The non-human manual harboring the mutative DNA of the inventionfeatures a high expression of the mutative DNA and, therefore, mayeventually develop adiaphoria associated with functional inactivation ofthe polypeptide of the invention through inhibition of the function ofthe endogenous normal DNA. Thus, the animal can be utilized as an animalmodel of that disease. For example, by using the DNA-transferred animalharboring the mutative DNA of the invention, analysis of the mechanismof this functional inactivation adiaphoria attributable to thepolypeptide of the invention and therapeutic modalities for the diseasecan be explored.

As a specific potential use, the DNA-transferred animal with a highexpression of the mutative DNA of the invention can be used as a modelfor elucidating the functional inhibition of the normal polypeptide bythe mutative polypeptide of the invention (dominant negative effect) inadiaphoria of functional inactivation.

Moreover, the DNA-transferred mammal harboring the foreign mutative DNAof the invention develops symptoms due to an increase in the freepolypeptide of the invention and, therefore, can be utilized in thescreening of therapeutic drugs for adiaphoria attributable to functionalinactivation of the polypeptide of the invention.

As other potential uses of the two types of DNA-transferred animalsharboring the two kinds of DNAs of the invention, the following may beconsidered:

-   (1) Use as a cell source for tissue culture;-   (2) Analysis of those genes or polypeptides which are expressed or    activated or deactivated specifically by the polypeptide of the    invention, by comparing and analyzing the DNA or RNA in tissues of    the DNA-transferred animal of the invention with the DNA or RNA of    non-DNA-transferred animal (control animal) or by comparing and    analyzing the compositions of the polypeptides expressed;-   (3) Study of the functions of cells of those tissues which are    generally difficult to culture, by using the cells from the tissues    containing the DNA as cultured by the standard tissue culture    technique;-   (4) Screening for drugs capable of enhancing the cell functions by    using the cells described in (3); and-   (5) Isolation and purification of the mutant polypeptide of the    invention and construction of antibodies thereto.

Furthermore, by using the DNA-transferred animal of the invention,clinical symptoms of diseases associated with the polypeptide of theinvention, inclusive of above-described adiaphoria associated withfunctional inactivation of the polypeptide of the invention, can beinvestigated. In addition, more detailed pathological findings can beobtained in various organs of this model of diseases associated with thepolypeptide of the invention, thus contributing to the development ofnew therapies as well as the study and treatment of secondary diseasesarising from such diseases.

Moreover, by removing various organs from the DNA-transferred animal ofthe invention, mincing them and digesting them with a proteolytic enzymesuch as trypsin, free single cells harboring the transferred DNA can berecovered. These cells can be cultured for establishment of a cell line.Furthermore, characterization of cells producing the polypeptide of theinvention can be made and their relationship with apoptosis,differentiation, or proliferation, the mechanism of signal transductionin them, and mutativeities involved can be explored to thereby generateinformation useful for further elucidation of the polypeptide of theinvention and its effects.

Moreover, for the development of therapeutic drugs for diseasesassociated with the polypeptide of the invention, such as adiaphoriaresulted from functional inactivation of the polypeptide of theinvention by using the DNA-transferred animal of the invention, aneffective and rapid screening technology for such therapeutic drugs canbe established by using the test and assay methods describedhereinbefore. In addition, by using the above DNA-transferred animal orthe foreign DNA expression vector of the invention, gene therapies fordiseases associated with the polypeptide of the invention can beexplored and developed.

(8) Knockout Animals

The invention further provides non-human-mammalian embryonic stem cellswherein the DNA of the invention is inactivated, and non-human mammalsdeficient in expression of the DNA of the invention wherein the DNA ofthe invention is deactivated.

The invention, therefore, provides:

-   (1) A non-human mammalian embryonic stem cell wherein the DNA of the    invention is inactivated;-   (2) The embryonic stem cell according to in (1) wherein the DNA is    inactivated by introduction of a reporter gene (e.g. E. coli-derived    β-galactosidase gene);-   (3) The embryonic stem cell according to (1) which is    neomycin-resistant;-   (4) The embryonic stem cell according to (1) wherein the non-human    mammal is a rodent;-   (5) The embryonic stem cell according to (4) wherein the rodent is    mouse;-   (6) A non-human mammal deficient in expression of the DNA of the    invention, wherein the DNA is inactivated;-   (7) The nonhuman mammal according to (6) wherein the DNA is    inactivated by introduction of a reporter gene (e.g. E coli-derived    β-galactosidase gene) and the reporter gene can be expressed under    the control of the promoter for the DNA of the invention;-   (8) The non-human mammal according to (6) wherein the non-human    mammal is a rodent;-   (9) The non-human mammal according to (8) wherein the rodent is    mouse; and-   (10) A method for screening for compounds, or salts thereof, that    enhance or inhibit the promoter activity for the DNA of the    invention, which comprises administering a test compound to the    non-human mammal according to (7) and detecting expression of the    reporter gene.

The expression “non-human mammalian embryonic stem cell wherein the DNAof the invention is inactivated” means the embryonic stem cell(hereinafter referred to briefly as ES cell) of a non-human mammal inwhich the DNA has been deprived of the capacity to express thepolypeptide of the invention (hereinafter, sometimes referred to as the“knockout DNA of the invention”) through introduction of an artificialmutation to the DNA of the invention possessed by the non-human manualto thereby inhibit expression of the DNA of the invention or throughsubstantial deprivation of the activity of the polypeptide of theinvention encoded by the DNA.

As the non-human mammals, the same animals as mentioned hereinbefore maybe used.

Examples of the method for introducing an artificial mutation to the DNAof the invention are a deletion of some or all of the DNA sequence, oran insertion of a different DNA, or substitution with a different DNA bythe genetic engineering technology. The knockout DNA of the inventionmay be created by such a mutation that would shift the reading frame ordestroy the function of the promoter or exon.

The non-human mammalian embryonic stem cell wherein the DNA of theinvention is inactivated (hereinafter referred to as the “DNAinactivated ES cell of the invention” or the “knockout ES cell of theinvention”) can be prepared by, for example, procedures which compriseisolating the DNA of the invention from the desired non-human mammal,inserting a drug-resistance gene, typically neomycin-resistance gene orhygromycin-resistance gene, or a reporter gene such as lacZ(β-galactosidase gene) or cat (chloramphenicol acetyltransferase gene)into its exon region to disrupt the function of the exon or inserting aDNA sequence for terminating genie transcription (e.g. poly A additionsignal) in an intron region between exons to thereby inhibit synthesisof a complete mRNA, introducing the thus-constructed DNA strand having aDNA sequence designed to eventually disrupt the gene (hereinafter,referred to briefly as the “targeting vector”) into the chromosomes ofthe host animal by homologous recombination, subjecting the resulting EScell to Southern hybridization analysis using a DNA sequence located onthe DNA of the invention or in its vicinity as a probe or a PCRprocedure using a DNA sequence located on the targeting vector and a DNAsequence in the vicinity but not including the DNA of the invention usedin the construction of the targeting vector as primers, and selectingthe knockout ES cell of the invention.

The original ES cell to be used for inactivation of the DNA of theinvention by the homologous recombination technique or the like may bean already established cell line such as those mentioned hereinbefore ora new cell line established de novo by the known method of Evans andKaufman. Taking mouse ES cells as an example, ES cells of the 129 lineare generally used but the immunological background of this line is notclear. Therefore, the alternative cell line for preparing pure-line EScells with an immunologically defined genetic background can be usedwith advantage, for example, BDF₁ mice created by the hybridization ofC57BL/6 mice and C57BL/6 mice, both yielding few eggs, with DBA/2 mice(BDF₁=F₁ of C57BL/6 and DBA/2). In addition to the advantage of high eggoutput and sturdiness of the egg, BDF₁ mice have the background ofC57BL/6 mice and so that in the construction of a disease model with EScells obtained, the genetic background of the model mice can beconverted to that of C57BL/6 mice by back-crossing with C57BL/6.

Moreover, in establishing an ES cell line, it is generally usedblastocytes 3.5 days following fertilization but, aside from them, alarge number of early embryos can be prepared with high efficiency byharvesting the embryos at the 8-cell stage and culturing them intoblastocytes.

Furthermore, while ES cells can be used from both male and femaleanimals, generally ES cells of male animals are more convenient for theconstruction of reproduction line chimeras. Moreover, for the purpose ofreducing the burden of the complicated cultural procedure, it ispreferable to carry out sexing as early as possible.

As a typical method for sexing ES cells, there can be mentioned themethod in which the gene in the sex determination region on the Ychromosome is amplified and detected by PCR. Whereas the conventionalkaryotype analysis requires about 10⁶ cells, the above method requiresonly about one colony equivalent of ES cells (about 50 cells).Therefore, the primary selection of ES cells in an early stage can bemade by this sexing method. Since male cells can thus be selected in theearly stage, the trouble in the initial stage of culture can bedrastically reduced.

Moreover, the secondary selection can be carried out by G-banding forthe number of chromosomes. The number of chromosomes in the resulting EScell is preferably 100% of the normal number but this goal may not bereached due to the physical and other factors involved in theestablishment of the line. In such cases, it is preferable to knockoutthe gene of the ES cell and re-clone it into the normal cell (taking amouse as an example, the cell in which the number of chromosomes is2n=40).

The embryonic stem cell line thus established is generally verysatisfactory in proliferation characteristic but since it is liable tolose its ontogenic ability, it must be subcultured with sufficient care.For example, this cell line should be cultured on suitable feeder cellssuch as STO fibroblasts in the presence of LIF (1–10000 U/ml) in acarbon dioxide incubator (preferably 5% CO₂, 95% air or 5% oxygen, 5%CO₂, 90% air) at about 37° C. And, in subculture, it should be treatedwith trypsin/EDTA solution (generally 0.001–0.5% trypsin/0.1–5 mM EDTA,preferably about 0.1% trypsin/1 mM EDTA) to provide single cells andseed them on freshly prepared feeder cells. While such subculture isgenerally performed every 1–3 days, it is good practice to observe thecells on each occasion and, whenever morphologically mutative cells arediscovered, discard the culture.

ES cells can be allowed to differentiate into various types of cells,such as head long muscle cells, visceral muscle cells, heart musclecells. etc. by conducting monolayer culture to a high density undersuitable conditions or suspension culture until a mass of cells isformed (M. J. Evans & M. H. Kaufman, Nature, 292, 154, 1981; G. R.Martin, Proceedings of National Academy of Science USA, 78, 7634, 1981;T. C. Doetschman et al., Journal of Embryology and ExperimentalMorphology, 87, 27, 1985), and the cell deficient in expression of theDNA of the invention as obtainable by causing the ES cell of theinvention to differentiate is useful for the cytobiological in vitrostudy of the polypeptide of the invention.

The nonhuman mammal deficient in expression of the DNA of the inventioncan be differentiated from normal animals by assaying the mRNA in theanimals by the known method and comparing the amounts of expressionindirectly.

As the non-human mammal used for this purpose, the same animals asmentioned hereinbefore may be used.

With respect to the non-human mammal deficient in expression of the DNAof the invention, the DNA of the invention can be knocked out byintroducing the targeting vector constructed as described above into,for example, mouse embryonic stem cells or mouse egg cells and therebyallowing the DNA sequence of the targeting vector harboring theinactivated DNA of the invention to undergo homologous recombinationwith, and accordingly replacing, the DNA of the invention on the mouseembryonic stem cell or egg cell chromosomes.

The cell with the DNA of the invention thus knocked out can be judged bySouthern hybridization analysis using a DNA sequence on the DNA of theinvention or in its vicinity as a probe or by PCR using a DNA sequenceon the targeting vector or a mouse-derived DNA sequence in a regionadjacent to but not including the DNA of the invention used in thetargeting vector as primers. When a non-human mammalian embryonic stemcell is used, a cell line with the DNA of the invention knocked out bythe homologous recombination technique is cloned and injected into thenon-human mammalian embryo or blastocyte at a suitable stage ofembryogenesis, for example at the 8-cell stage, and the resultingchimera embryo is transplanted in the pseudopregnant uterus of thenon-human mammal. The animal thus obtained is a chimera animalconstituted by both the cells harboring the normal DNA locus of theinvention and the cells harboring the artificially mutated DNA locus ofthe invention.

When some of the gametes of this chimera animal harbor the mutated DNAlocus of the invention, an individual whose entire tissue is constitutedby cells harboring the mutated DNA locus of the invention can bescreened from the colony of animals obtained by crossing such a chimeraanimal with a normal animal, for example by coat color discrimination.The individuals thus selected are usually animals hetero-deficient inexpression of the polypeptide of the invention and by mating suchindividuals hetero-deficient in expression of the polypeptide of theinvention with each other, animals homo-deficient in expression of thepolypeptide of the invention can be acquired.

When egg cells are used, a transgenic non-human mammal with thetargeting vector having been introduced into its chromosomes can beprepared by injecting the DNA solution into the egg cell nucleus by themicroinjection technique and selecting animals expressing a mutation ofthe DNA of the invention by homologous recombination.

The individuals with the DNA of the invention thus knocked out are matedto verify that the animals obtained by mating also have the DNA knockedout and they can be sub-bred under the usual breeding conditions.

Preparation and maintenance of the germ line may also be carried out inaccordance with conventional methods. Thus, by mating male and femaleanimals harboring the inactivated DNA, homozygotes having theinactivated DNA in both homologous chromosomes can be obtained. Thehomozygotes thus obtained are bred under such conditions that, withregard to the dam, the number of homozygotes is plural per normalindividual. By mating male and female heterozygotes, homozygotes andheterozygotes both harboring the inactivated DNA can be sub-bread.

The non-human mammalian embryonic stem cell harboring the inactivatedDNA of the invention is very useful for the construction of non-humanmammals deficient in expression of the DNA of the invention.

Moreover, the mammal deficient in expression of the polypeptide of theinvention lacks the various biological activities inducible by thepolypeptide of the invention and can, therefore, be of use as an animalmodel of diseases arising from inactivation of the biological activitiesof the polypeptide of the invention, thus being useful in theetiological studies of such diseases and development of therapeuticmethods.

(8a) Method for Screening for Compounds with Therapeutic/ProphylacticEffect upon Diseases Resulted from Deficiency of or Damage to the DNA ofthe Invention

Non-human mammals deficient in expression of the DNA of the inventionmay be used for screening for compounds with a therapeutic and/orprophylactic effect upon diseases resulted from deficiency of or damageto the DNA of the invention, for example, diseases accompanied byneurodegeneration, such as neurodegenerative diseases [e.g. Alzheimer'sdisease (familial Alzheimer's disease, juvenile Alzheimer's disease,sporadic Alzheimer's disease, etc.), Parkinson's disease, Down syndrome,amyotrophic lateral sclerosis, prion disease, Creutzfeldt-Jacob disease,Huntington's chorea, diabetic neuropathy, multiple sclerosis, etc.],brain dysfunctions (e.g. brain infarction, cerebral hemorrhage,subarachnoid hemorrhage, ischemic brain diseases, epidural hematoma,subdural hematoma, etc.), cancers (e.g. astrocytoma, oligodendroglioma,etc.), immunological diseases, infections (e.g. meningitis,protozoiasis, rickettsial infections, metazoan infections, bacterial orviral meningitis such as Borna's disease, postvaccinal encephalitis,AIDS encephalopathy, etc.), gastrointestinal diseases, circulatorydiseases and endocrine diseases.

The present invention provides a method for screening for compounds, orsalts thereof, having a therapeutic and/or prophylactic effect upondiseases resulted from deficiency of or damage to the DNA of theinvention, which is characterized by administering a test compound to anon-human mammal deficient in expression of the DNA of the invention aidobserving and measuring the changes in the mammal.

As the nonhuman mammal deficient in expression of the DNA of theinvention, the same animals as described earlier may be used.

The test compound may be, for example, a peptide, protein, non-peptidiccompound, synthetic compound, fermentation product, cell extract, plantextract, animal tissue extract or plasma. These compounds may be eithernovel compounds or known compounds.

Specifically, a non-human mammal deficient in expression of the DNA ofthe invention is treated with a test compound and then compared with acontrol animal not treated with the compound. Subsequently, thetherapeutic and/or prophylactic effect of the test compound may beexamined using the changes in individual organs, tissues or diseasesymptoms in the mammal as indicators.

As a method for treating a test animal with a test compound, oraladministration, intravenous injection, or the like may be used. Themethod may be appropriately selected depending on the symptoms of thetest animal, the nature of the test compound, and so on. Dose levels ofthe test compound may be appropriately selected taking into account ofthe administration method, the nature of the test compound, and so on.

Compounds obtainable by using the screening method of the invention arecompounds selected from the above-mentioned test compounds, and have atherapeutic and/or prophylactic effect upon diseases resulted fromdeficiency of or damage to the DNA of the invention, for example,diseases accompanied by neurodegeneration, such as neurodegenerativediseases [e.g. Alzheimer 's disease (familial Alzheimer's disease,juvenile Alzheimer's disease, sporadic Alzheimer's disease, etc.),Parkinson's disease, Down syndrome, amyotrophic lateral sclerosis, priondisease, Creutzfeldt-Jacob disease, Huntington's chorea, diabeticneuropathy, multiple sclerosis, etc.], brain dysfunctions (e.g. braininfarction, cerebral hemorrhage, subarachnoid hemorrhage, ischemic braindiseases, epidural hematoma, subdural hematoma, etc.), cancers (e.g.astrocytoma, oligodendroglioma, etc.), immunological diseases,infections (e.g. meningitis, protozoiasis, rickettsial infections,metazoan infections, bacterial or viral meningitis such as Borna'sdisease, postvaccinal encephalitis, AIDS encephalopathy, etc.),gastrointestinal diseases, circulatory diseases and endocrine diseases.Therefore, they may be used as medicines that are safe and of lowtoxicity, for example as prophylactic and/or therapeutic agents forthose diseases that are safe and of low toxicity. Furthermore, compoundsinducible from those compounds obtained by the above screening may alsobe used in the same manner.

The compound obtained by the above screening may be in a salt form. Assalts of the compounds, salts formed with physiologically acceptableacids (e.g. organic or inorganic acids) or bases (e.g. alkali metals)may be used. Especially preferable are physiologically acceptable acidaddition salts. Examples of such salts include salts formed withinorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromicacid or sulfuric acid) and salts formed with organic acids (e.g. aceticacid, formic acid, propionic acid, fumaric acid, maleic acid, succinicacid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid or benzenesulfonic acid).

Medicines comprising the compound, or a salt thereof, obtained by thescreening may be prepared in the same manner as described for medicinescomprising the polypeptide of the invention.

Since the thus obtained preparations are safe and of low toxicity, theymay be administered to, for example, mammals (such as human, rat, mouse,guinea pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).

Dose levels of the above compound or a salt thereof may vary dependingupon the target disease, the patient to be treated, administrationroute, and so on. When the compound is administered orally for treatingAlzheimer's disease, generally the compound is administered to adultpatients (60 kg in body weight) at a dose of about 0.1–100 mg/day,preferably about 1.0–50 mg/day, more preferably about 1.0–20 mg/day.With respect to parenteral administration, when the compound isadministered to adult patients (60 kg in body weight) in the form of aninjection for treating Alzheimer's disease, it is convenient tointravenously inject the compound at a dose of about 0.01–30 mg/day,preferably about 0.1–20 mg/day, and more preferably about 0.1–10 mg/day,though the dose per administration may vary depending on the patient tobe treated, the target disease, etc. For other animals, correspondingdoses may be administered after conversion of the above-mentioned valuesper 60 kg based on actual body weights.

(8b) Method for Screening for Compounds that Promote or Inhibit PromoterActivity for the DNA of the Invention

The present invention provides a method for screening for compounds, orsalts thereof, that promote or inhibit promoter activity for the DNA ofthe invention, which is characterized by administering a test compoundto a non-human mammal deficient in expression of the DNA of theinvention and detecting the expression of a reporter gene.

In the above screening method, there is used a non-human mammaldeficient in expression of the DNA of the invention wherein the DNA ofthe invention is inactivated as a result of introduction of a reportergene, and this reporter gene is capable of being expressed under thecontrol of the promoter for the DNA of the invention.

As the test compound, the compounds as enumerated above may be used.

As the reporter gene, the genes as enumerated above may be used. Amongall, β-galactosidase gene (lacZ), soluble alkali phosphatase gene orluciferase gene may be preferably used.

In the non-human mammal deficient in expression of the DNA of theinvention wherein the DNA of the invention is replaced with a reportergene, since the reporter gene is present under the control of thepromoter for the DNA of the invention, the promoter activity can bedetected by tracing the expression of the substance encoded by thereporter gene.

For example, when a part of the DNA region encoding the polypeptide ofthe invention is replaced with E. coli-derived β-galactosidase gene(lacZ), β-galactosidase is expressed instead of the polypeptide of theinvention in those tissues where originally the polypeptide of theinvention has been expressed. Thus, by staining with a substrate forβ-galactosidase such as5-bromo-4-chloro-3-indolyl-β-D-galactopyrasosidase (X-gal), it ispossible to observe the state of in vivo expression of the polypeptideof the invention in the mammal simply. Specifically, nice deficient inthe polypeptide of the invention or tissue sections thereof may be fixedin glutaraldehyde or the like, washed with phosphate-bufferedphysiological saline (PBS), and treated with a staining solutioncontaining X-gal at room temperature or around 37° C. for about 30 minto 1 hr. Subsequently, the tissue samples are washed with 1 mM EDTA/PBSsolution to terminate the β-galactosidase reaction, followed byobservation of the resultant color development. Alternatively, mRNAencoding lacZ may be detected according to conventional methods.

The compounds or salts thereof obtainable by the above-describedscreening are compounds that are selected from the above-mentioned testcompounds and yet promote or inhibit the promoter activity for the DNAof the invention.

The compound obtained by the above screening may be in a salt form. Assalts of the compound, salts formed with physiologically acceptableacids (e.g. organic or inorganic acids) or bases (e.g. alkali metals)may be used. Especially preferable are physiologically acceptable acidaddition salts. Examples of such salts include salts formed withinorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromicacid or sulfuric acid) and salts formed with organic acids (e.g. aceticacid, formic acid, propionic acid, fumaric acid, maleic acid, succinicacid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid or benzenesulfonic acid).

Since compounds, or salts thereof, that promote the promoter activityfor the DNA of the invention can promote the expression of thepolypeptide of the invention and thereby promote the function thereof,they are useful as medicines, for example, as prophylactic and/ortherapeutic agents for diseases accompanied by neurodegeneration, suchas neurodegenerative diseases [e.g. Alzheimer's disease (familialAlzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer'sdisease, etc.), Parkinson's disease, Down syndrome, amyotrophic lateralsclerosis, prion disease, Creutzfeldt-Jacob disease, Huntington'schorea, diabetic neuropathy, multiple sclerosis, etc.], braindysfunctions (e.g. brain infarction, cerebral hemorrhage, subarachnoidhemorrhage, ischemic brain diseases, epidural hematoma, subduralhematoma, etc.), cancers (e.g. astrocytoma, oligodendroglioma, etc.),immunological diseases, infections (e.g. meningitis, protozoiasis,rickettsial infections, metazoan infections, bacterial or viralmeningitis such as Borna's disease, postvaccinal encephalitis, AIDSencephalopathy, etc.), gastrointestinal diseases, circulatory diseasesand endocrine diseases. More preferably, they are useful as prophylacticand/or therapeutic agents for Alzheimer's disease.

Further, those compounds inducible from the compounds obtained from theabove screening may also be used in the same manner.

Medicines comprising the compound or, a salt thereof, obtained by thescreening may be prepared in the same manner as described for medicinescomprising the polypeptide of the invention or a salt thereof.

Since the thus obtained preparations are safe and of low toxicity, theymay be administered to, for example, manuals (such as rat, human, mouse,guinea pig, rabbit, sheep, pig, bovine, horse, cat, dog, monkey, etc.).

Dose levels of the above compound or a salt thereof may vary dependingupon the target disease, the patient to be treated, administrationroute, and so on. When a compound that promotes the promoter activityfor the DNA of the invention is administered orally for treatingAlzheimer's disease, generally the compound is administered to adultpatients (60 kg in body weight) at a dose of about 0.1–100 mg/day,preferably about 1.0–50 mg/day, more preferably about 1.0–20 mg/day.With respect to parenteral administration, when a compound that promotesthe promoter activity for the DNA of the invention is administered toadult patients (60 kg in body weight) in the form of an injection fortreating Alzheimer's disease, it is convenient to intravenously injectthe compound at a dose of about 0.01–30 mg/day, preferably about 0.1–20mg/day, and more preferably about 0.1–10 mg/day, though the dose peradministration may vary depending on the patient to be treated, thetarget disease, etc. For other animals, corresponding doses may beadministered after conversion of the above-mentioned values per 60 kgbased on actual body weights.

On the other hand, when a compound that inhibits the promoter activityfor the DNA of the invention is administered orally, generally thecompound is administered to adult patients with Alzheimer's disease (60kg in body weight) at a dose of about 0.1–100 mg/day, preferably about1.0–50 mg/day, more preferably about 1.0–20 mg/day. With respect toparenteral administration, when a compound that inhibits the promoteractivity for the DNA of the invention is administered to adult patientswith Alzheimer's disease (60 kg in body weight) in the form of aninjection, it is convenient to intravenously inject the compound at adose of about 0.01–30 mg/day, preferably about 0.1–20 mg/day, and morepreferably about 0.1–10 mg/day, though the dose per administration mayvary depending on the patient to be treated, the target disease, etc.For other animals, corresponding doses may be administered afterconversion of the above-mentioned values per 60 kg based on actual bodyweights.

Thus, the non-human mammal deficient in expression of the DNA of theinvention is extremely useful in screening for compounds, or saltsthereof, that promote or inhibit the promoter activity for the DNA ofthe invention, and may contribute greatly to the elucidation of causesof various diseases resulted from deficiency in expression of the DNA ofthe invention, for example, diseases accompanied by neurodegeneration,such as neurodegenerative diseases [e.g. Alzheimer's disease (familialAlzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer'sdisease, etc.), Parkinson's disease, Down syndrome, amyotrophic lateralsclerosis, prion disease, Creutzfeldt-Jacob disease, Huntington'schorea, diabetic neuropathy, multiple sclerosis, etc.], braindysfunctions (e.g. brain infarction, cerebral hemorrhage, subarachnoidhemorrhage, ischemic brain diseases, epidural hematoma, subduralhematoma, etc.), cancers (e.g. astocytoma, oligodendroglioma, etc.),immunological diseases, infections (e.g. meningitis, protozoiasis,rickettsial infections, metazoan infections, bacterial or viralmeningitis such as Borna's disease, postvaccinal encephalitis, AIDSencephalopathy, etc.), gastrointestinal diseases, circulatory diseasesand endocrine diseases, or to the development of prophylactic and/ortherapeutic agents for such diseases.

In the specification and drawings of the present application, theabbreviations used for bases (nucleotides), amino acids and so forth arethose recommended by the IUPAC-IUB Commission on BiochemicalNomenclature or those conventionally used in the art. Examples of suchabbreviations are given below. Amino acids that may have optical isomersare intended to represent their L-isomer unless otherwise specified.

-   DNA: Deoxyribonucleic acid-   cDNA: Complementary deoxyribonucleic acid

A: Adenine

T: Thymine

G: Guanine

C: Cytosine

RNA: Ribonucleic acid

mRNA: Messenger ribonucleic acid

dATP: Deoxyadenosine triphosphate

dTTP: Deoxythymidine triphosphate

dGTP: Deoxyguanosine triphosphate

dCTP: Deoxycytidine triphosphate

ATP: Adenosine triphosphate

EDTA: Ethylenediaminetetracetic acid

SDS: Sodium dodecyl sulfate

Gly: Glycine

Ala: Alanine

Val: Valine

Leu: Leucine

Ile: Isoleucine

Ser: Serine

Thr: Threonine

Cys: Cysteine

Met: Methionine

Glu: Glutamic acid

Asp: Aspartic acid

Lys: Lysine

Arg: Arginine

His: Histidine

Phe: Phenylalanine

Tyr: Tyrosine

Trp: Tryptophan

Pro: Proline

Asn: Asparagine

Gln: Glutamine

pGlu: Pyroglutamic acid

The substituents, protective groups and reagents which are frequentlyused in the specification are represented by the followingabbreviations.

Me: Methyl

Et: Ethyl

Bu: Butyl

Ph: Phenyl

TC: Thiazolidine-4(R)-carboxamide

Tos: p-Toluene sulfonyl

CHO: Formyl

Bzl: Benzyl

Cl₂-Bzl: 2,6-Dichlorobenzyl

Bom: Benzyloxymethyl

Z: Benzyloxycarbonyl

Cl-Z: 2-Chlorobenzyloxycarbonyl

Br-Z: 2-Bromobenzyloxycarbonyl

Boc: t-Butoxycarbonyl

DNP: Dinitrophenol

Trt: Trityl

Bum: t-Butoxymethyl

Fmoc: N-9-Fluorenylmethyloxycarbonyl

HOBt: 1-Hydroxybenzotriazole

HOOBt: 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine

HONB: 1-Hydroxy-5-norbornene-2,3-dicarboximide

DCC: N,N′-Dicyclohexylcarbodiimide

Pbf: 2,2,4,6,7-Pentanethyldihydrobenzofuran-5-sulfonyl

tBu: Tertiary butyl

TFA: Trifluoroacetate

OHAt: 1-Hydroxy-7-azabenzotriazol

PyAop: 7-Azabenzotriazole-1-yloxytris(pyrrolidino)phosphoniumhexafluorophosphate

DIPCDI: 1,3-Diisopropyl-carbodiimide

Fmoc-Leu-Ser(Psi(Me,Me)pro)-OH:(4S)-3-(Fmoc-Leu)-2,2,-dimethyloxazolidine-4-carboxylic acid)]

The SEQ ID NOS of the SEQUENCE LISTING of the present specificationrepresent the sequences as indicated below.

-   [SEQ ID NO: 1]

This shows the nucleotide sequence of a primer used in Example 1.

-   [SEQ ID NO: 2]

This shows the nucleotide sequence of a primer used in Example 1.

-   [SEQ ID NO: 3]

This shows the nucleotide sequence of a DNA encoding the polypeptide ofthe invention obtained in Example 1.

-   [SEQ ID NO: 4]

This shows the amino acid sequence of the polypeptide of the inventionobtained in Example 1.

-   [SEQ ID NO: 5]

This shows the nucleotide sequence of a query used in Example 1.

-   [SEQ ID NO: 6]

This shows the nucleotide sequence of the DNA obtained in Example 1comprising the SEQ ID NO: 3.

-   [SEQ ID NO: 7]

This shows the amino acid sequence of a partial peptide of thepolypeptide of the invention (SEQ ID NO: 4) obtained in Example 1.

-   [SEQ ID NO: 8]

This shows the nucleotide sequence of a DNA encoding SEQ ID NO: 7.

Transformant Escherichia coli TOP10/pcDNA-hn3 obtained in Example 1described later has been deposited with the International PatentOrganism Depository, National Institute of Advanced Industrial Scienceand Technology located at Central 6, 1-1 Higashi 1-chome, Tsukuba City,Ibaraki Pref. (zip code No.: 305-8566) since Jul. 19, 2001 wider theAccession No. FERM BP-7674 and with the Institute for Fermentation,Osaka (IFO) located at 17–85, jusanbonncho 2-chome, Yodogawa-ku, OsakaCity, Osaka Pref. (zip code No.:532–8686) since Jul. 3, 2001 under theAccession No. IFO 16673.

EXAMPLES

Hereinbelow, the present invention will be described more specificallywith reference to the following Examples. However, the present inventionis not limited to these Examples. Genetic manipulations using E. coliwere carried out in accordance with the methods described in the book“Molecular Cloning”.

Example 1

Database GEMBLE was searched for using the human humanin-like genecoding region as a query. As a result, it was found that a humanin-likegene region comprising a start codon and a termination codon bothcorresponding to the coding region of the humanin gene is present withinthe sequence of Accession No. AL356135. In order to confirm that thisgene exists actually and that this gene is transcribed and functions,the inventors have prepared a cDNA by reverse transcription using 1.0 μgof human total brain poly A⁺ RNA (Clontech) as a template, SuperScriptreverse transcriptase (Gibco BRL) and, according to the manual attachedto the transcriptase, Oligo (dT) primers. Then, two primersTACCCTAACCGTGCAAAGGTAGCATG (SEQ ID NO: 1) andGTGGGCTTATTGGGTGTTGTTTGCATTGG (SEQ ID NO: 2) located upstream of 5′ endand downstream of 3′ end, respectively, of the humanin-like sequence inAccession No. AL356135 sequence were designed, followed by a PCR in 20μl of a reaction solution. This reaction solution was composed of thecDNA solution as a template in an amount equivalent to 10 ng of mRNA,0.5 μM each of the primers, 2.5 mM MgCl₂, 0.2 mM dNTP, 1/100 volume ofAmpliTaq Gold (Perkin Elmer), and 1/10 vlume of 10× AmpliTaq GoldBuffer. Following the first denaturation at 95° C. for 10 min, 40 cyclesof at 95° C. for 15 sec, at 67° C. for 15 sec and at 72° C. for 15 secwere performed, followed by final extension at 72° C. for 5 min. Theamplified DNA in the resultant solution was sub-cloned into plasmidvector pcDNA3.1/V5/His-TOPO using Eukaryotic TOPO TA cloning kit(Invitrogen) and then introduced into E. coli TOP10. From the resultanttransformant, plasmid DNA was purified using QIA prep8 mini prep(Qiagen). Reactions for determining the nucleotide sequence wereperformed using BigDye Terminator Cycle Sequence Ready Reaction Kit(Perkin Elmer) and analyzed with an automated fluorescence sequencer. Asa result, a nucleotide sequence as shown in SEQ ID NO: 6 was obtainedwhich comprises the coding region of the humanin-like sequence (SEQ IDNO: 3) found in the above-described search. Thus, it was confirmed thatthis gene is expressed in human total brain.

Since this sequence (SEQ ID NO: 6) comprises the full length of thecoding region of the humanin-like sequence, E. coli TOP10 wastransformed with the above-described plasmid to thereby obtain ofEscherichia coli TOP 10/pcDNA-hn3.

Example 2

A polypeptide comprising the amino acid sequence as shown in SEQ ID NO:4 is sometimes referred to as a “humanin-like peptide”.

A humanin-like peptide was prepared as described below.

Briefly, 0.25 mmol of Fmoc-Thr(tBu)-O-Clt resin (0.527 mmol/g) [whereinFmoc-Thr(tBu)-OH is introduced into a commercial 2-chlorotrityl resin(Clt resin, 1.33 mmol/g)] was placed in the reactor of a peptidesynthesizer ABI 433A, and solid-phase peptide synthesis was performed bythe Fmoc/DCC/HOBt method. As the side-chain protective groups for Fmocamino acids, Pbf group was used for Arg; Boc group was used for Lys; tBugroup was used for Asp, Thr and Ser; and Trt group was used for Cys.Other amino acids were used without side-chain protective groups. Thepeptide chain was extended to position 13 of the above-mentionedsequence (Thr). To the resultant Fmoc-humanin-like peptide (13-24)-0-Cltresin, Fmoc-Leu-Ser(Psi(Me,Me)pro)-OH (NOVA; product No. 05-20-1004) wasintroduced with DIPCDI/HOAt. Then, amino acids were introduced in orderfrom Leu at position 10 toward the N-terminal with DCC/HOBt, to therebyobtain a protected peptide resin of interest.

This resin (100 mg) was agitated in a mixed solution (total 1.5 ml) ofTFA, thioanisole, m-cresol, water, triisopropylsilane and ethanidithiol(80:5:5:5:2.5:2.5) at room temperature for 1.5 hrs. Then, ether wasadded to the reaction solution to deposit white powder. The reactionsolution was centrifuged, followed by removal of the supernatant. Theseoperations were repeated three times. The resultant residue wasextracted with water and then lyophilized to obtain white powder. Thethus obtained crude peptide was subjected to preparative HPLC using YMCPack R&D-ODS-5-B S-5, 120A column (30×250 nm). A linear-typeconcentration gradient elution (60 min) was performed using mobile phaseA (0.1% TFA in water) and mobile phase B (0.1% TFA in acetonitrile) atA/B ratios of from 78/22 to 68/32. Fractions containing the peptide ofinterest were collected and lyophilized to obtain 21.8 mg of whitepowder.

ESI-MS: M⁺ 2692.8 (theoretical value: 2692.5)

HPLC elution time: 10.5 min

Elution conditions:

Column YMC AM 301 (4.6×100 mm)

Mobile phase A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile

A/B: from 80/20 to 30/70, linear-type concentration gradient elution (25min)

Flow rate: 1.0 ml/min

Example 3

Inhibitory Activity of the Humanin-Like Peptide against GlutanicAcid-Induced Cell Death of Rat Adrenal Medulla-Derived PheochromocytomaCell PC12h

PC12h cells (supplied by Prof. Hiroshi Hatanaka, Institute for ProteinResearch, Osaka University; Hatanaka, H., Brain Research 222: 225–233,1981) were plated at a density of 2×10⁴ cells/cm² in collagen-coated96-well plates (Iwaki) containing Dulbecco's modified Eagle's medium(hereinafter referred to as “DMEM”) containing 10% fetal bovine serumand 5% horse serum. After 24 hrs, the medium was exchanged for 100 μl ofDMEM containing 20 nM HEPES (pH 7.5). At the same time, variousconcentrations of the humanin-like peptide prepared in Example 2 (SEQ IDNO: 4) and glutamic acid to give a concentration of 1 mM were added tothe medium. After 72 hrs, cells were lysed with 100 μl of phosphatebuffered saline containing 0.2% Tween 20. Then, the lactatedehydrogenase (LDH) activity of the cell extract was determined with LDHCytotoxic Test WAKO (Wako Purechemical Industries).

The results are shown in FIG. 1.

Seventy-two hours after the glutamic acid treatment, while cell survivalratio was 34.0% in the humanin-like peptide not-added plot, the survivalratios were improved to 59.3% and 74.6%, respectively, by the additionof the humanin-like peptide at-1 μM and 10 μM. Here, cell survivalratios are expressed taking the survival in the glutamic acid non-addedplot as 100%.

From these results, it is clear that glutamic acid-induced cell death ofrat adrenal medulla-derived pheochromocytoma cell PC12h is inhibited bythe humanin-like peptide.

Example 4

Preparation of Another Humanin-Like Peptide (19–24) (SEQ ID NO: 7):Pro-Val-Lys-Arg-Arg-Thr

A protected peptide resin having the sequence of interest was preparedusing Fmoc-Thr(tBu)-O-Clt resin, and purified in the same manner asdescribed for the humanin-like peptide prepared in Example 3. As aresult, 29 mg of white powder was obtained.

ESI-MS: M⁺ 756.5 (theoretical value: 756.5)

HPLC elution time: 10.2 min

Elution conditions:

Column YMC AM 301 (4.6×100 mm)

Mobile phase A: 0.1% TFA in water; B: 0.1% TFA in acetonitrile

A/B: from 80/20 to 30/70, linear-type concentration gradient elution (25min)

Flow rate: 1.0 ml/min

INDUSTRIAL APPLICABILITY

The polypeptide and the polynucleotide of the invention can be used as adiagnostic, therapeutic and/or prophylactic agent for various diseasesincluding diseases accompanied by neurodegeneration, such asneurodegenerative diseases [e.g. Alzheimer's disease (familialAlzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer'sdisease, etc.), Parkinson's disease, Down syndrome, amyotrophic lateralsclerosis, prion disease, Creutzfeldt-Jacob disease, Huntington'schorea, diabetic neuropathy, multiple sclerosis, etc.], braindysfunctions (e.g. brain infarction, cerebral hemorrhage, subarachloidhemorrhage, ischemic brain diseases, epidural hematoma, subduralhematoma, etc.), cancers (e.g. astrocytoma, oligodendroglioma, etc.),immunological diseases, infections (e.g. meningitis, protozoiasis,rickettsial infections, metazoan infections, bacterial or viralmeningitis such as Borna's disease, postvaccinal encephalitis, AIDSencephalopathy, etc.), gastrointestinal diseases, circulatory diseasesand endocrine diseases; or as a cell death inhibitor. The polypeptide ofthe invention is also useful as a reagent for screening for thosecompounds or salts thereof which promote or inhibit the activity of thepolypeptide of the invention. Further, since antibodies to thepolypeptide of the invention can recognize the polypeptide of theinvention specifically, they can be used in the detection, quantitativedetermination or neutralization of the polypeptide of the invention insample solutions, and are useful in the diagnosis of various diseasesincluding diseases accompanied by neurodegeneration, such asneurodegenerative diseases [e.g. Alzheimer's disease (familialAlzheimer's disease, juvenile Alzheimer's disease, sporadic Alzheimer'sdisease, etc.), Parkinson's disease, Down syndrome, amyotroplic lateralsclerosis, prion disease, Creutzfeldt-Jacob disease. Huntington'schorea, diabetic neuropathy, multiple sclerosis, etc.], braindysfunctions (e.g. brain infarction, cerebral hemorrhage, subarachnoidhemorrhage, ischemic brain diseases, epidural hematoma, subduralhematoma, etc.), cancers (e.g. astrocytoma, oligodendroglioma, etc.),immunological diseases, infections (e.g. meningitis, protozoiasis,rickettsial infections, metazoan infections, bacterial or viralmeningitis such as Borna's disease, postvaccinal encephalitis, AIDSencephalopathy, etc.), gastrointestinal diseases, circulatory diseasesand endocrine diseases.

1. A polypeptide comprising an amino acid sequence as shown in SEQ IDNO: 4, or an amide, ester or salt thereof.
 2. The polypeptide accordingto claim 1 consisting of the amino acid sequence as shown in SEQ ID NO:4, or an amide, ester or salt thereof.
 3. A method for producing thepolypeptide according to claim 1 or an amide, ester or salt thereof,comprising culturing a transformant transformed with a recombinantvector comprising a polynucleotide having a nucleotide sequence encodingthe polypeptide according to claim 1; and allowing the polypeptideaccording to claim 1 to be produced and accumulated.
 4. A method forscreening for compounds, or salts thereof, that promote or inhibit theactivity of the polypeptide according to claim 1 or an amide, ester orsalt thereof, wherein the method is characterized by using thepolypeptide according to claim 1 or an amide, ester or salt thereof. 5.A kit for screening for compounds, or salts thereof, that promote orinhibit the activity of the polypeptide according to claim 1 or anamide, ester or salt thereof, which comprises the polypeptide accordingto claim 1 or an amide, ester or salt thereof.
 6. A medicine comprisingthe polypeptide according to claim 1 or an amide, ester or salt thereof.7. The medicine according to claim 6, wherein said medicine is aprophylactic and/or therapeutic agent for neurodegenerative disorders orbrain dysfunctions.
 8. The medicine according to claim 7, wherein saidmedicine is a prophylactic and/or therapeutic agent for Alzheimer'sdisease, Parkinson's disease, Down syndrome, amyotrophic lateralsclerosis, prion disease, Creutzfeldt-Jacob disease, Huntington'schorea, diabetic neuropathy, multiple sclerosis, brain infarction,cerebral hemorrhage, subarachnoid hemorrhage, ischemic brain diseases,epidural hematoma or subdural hematoma.
 9. The medicine according toclaim 7, wherein said medicine is a prophylactic and/or therapeuticagent for Alzheimer's disease.
 10. The medicine according to claim 6,wherein said medicine is a cell death inhibitor.
 11. A method ofpreventing and/or treating neurodegenerative diseases or braindysfunctions, which is characterized by administering to a mammal aneffective amount of the polypeptide according to claim 1 or an amide,ester or salt thereof.